Jeremiah Oshiomame Unuofin

Pharmacological investigations of Kedrostis africana (L.) Cogn. and Vernonia mespilifolia Less. used in folk medicine in the Eastern Cape, South Africa

JEREMIAH OSHIOMAME UNUOFIN

DEPARTMENT OF BOTANY FACULTY OF SCIENCE AND AGRICULTURE UNIVERSITY OF FORT HARE ALICE 5700, SOUTH AFRICA

MAY, 2017

Pharmacological investigations of Kedrostis africana (L.) Cogn. and Vernonia mespilifolia Less. used in folk medicine in the Eastern Cape, South Africa

JEREMIAH OSHIOMAME UNUOFIN

A thesis submitted in fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY IN ETHNOBOTANY

DEPARTMENT OF BOTANY FACULTY OF SCIENCE AND AGRICULTURE UNIVERSITY OF FORT HARE, ALICE SOUTH OF AFRICA

SUPERVISOR: PROF ANTHONY JIDE AFOLAYAN CO-SUPERVISOR: DR GLORIA ADERONKE OTUNOLA

MAY, 2017

DECLARATION

I, Jeremiah Oshiomame Unuofin, declare that this thesis, submitted to the University of Fort

Hare for the degree of Doctor of Philosophy in Ethnobotany in the Faculty of Science and

Agriculture, is my own work; and that this work has not been submitted to any other institution for the award of any academic degree. I declare that I followed the rules and conventions concerning referencing and citation in scientific writing. I also declare that all sources of materials used for this thesis have been duly acknowledged and accurately referenced. Again,

I declare that I am fully aware of the University of Fort Hare policy on plagiarism and I have taken every precaution to comply with the regulations of the University.

Name: Jeremiah Oshiomame Unuofin Signature:......

We confirm that the work reported here was carried out by the above named candidate under our supervision.

Prof Anthony Jide Afolayan

Signature: …………………………. Date: …………………………

Dr Gloria Aderonke Otunola

Signature: ...... Date: ......

DEDICATION

This work is dedicated to Almighty God, my family and whoever has a strong craving for research.

ACKNOWLEDGEMENTS

A dissertation cannot be completed alone. It would be impossible to acknowledge each individual that has contributed in some way. My profound gratitude goes to my Supervisor,

Prof Anthony J Afolayan for his advice and guidance throughout the course of my study. To my Co-Supervisor, Dr (Mrs) Gloria A Otunola, for all her contributions, advice and encouragement during the course of this study. I wish to appreciate all the Medicinal Plants and Economic Development (MPED) Research Centre past and present group members.

Special thanks to Dr Olubunmi Wintola, Dr Wilfred Mbeng, Dr Josephine Sharaibi, Dr

Callistus Bvenura, Dr Emmanuel Ajayi, Dr Samuel Odeyemi, Dr Ggenga Adeogun, Maureen

Mangoale, Bosede Elizabeth Famewo, Mrs Funke Paulina Adijat Ogundola, Vuyokasi

Mgobozi, Zimasa Dubeni, Dr Leye Akinleye, Dr Jonas Sagbo, Dr Linda Sowunmi, Yanga

Mhlomi, Siyasanga Mnciva, Zomsa Yako, Dr Ogochukwu and Taiwo Funmi. I convey my heartfelt thanks to my past and present office mates: Dr Sinbad Olorunnisola, Dr Cromwell

Kibiti, Dr Mojisola Asowata, Franklin Ohikhena, Funmi Adegbaju, Muhali Jimoh and Tomi

Lois Olatunji. I appreciate you all.

I wish to say a big thank you to my uncle Frank Unuofin (PhD) for his assistance in my admission process and encouragement during the course of my study.

“Friends in need are friends indeed”, I am really thankful and grateful to my sincere Pals

Feyisayo Ogunrinde, Yonda Fasae, Siwaphiwe Peteni, Ibitola Asaolu, Sandra Oloketuyi and

Joel Italhume for their constant encouragement and moral support throughout my programme.

I also appreciate my fellowship members at Deeper Life Campus Fellowship, Alice Campus for their love, support and encouragement through this journey.

I am grateful to my parents Mr Peter and Mrs Joy Unuofin for their unflinching love, care, support, prayers and words of advice and encouragement from the inception of my PhD

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programme. I salute the efforts of my siblings Victor, Christiana, John, Divine, my in-laws

(Austin and Janet) and my nephew Dominion and Dunamis in making this work a success.

Finally, I am deeply indebted to all animals whose precious lives were sacrificed during this research work.

ETHICAL APPROVAL FOR THE STUDY

A portion of this study involved the use of animals and was carried out following the approval of the University of Fort Hare’s Ethics Committee with protocol number AFO051SUNU01.

ABSTRACT

The prevalence of obesity and its co-morbidity is increasing in South Africa. High calorie diet, sedentary lifestyle and the cultural belief that being fat or obese signifies beauty, wellness and wealth are major causative factors. This study was undertaken to scientifically validate two major medicinal plants used traditionally in the Raymond Mhlaba Municipality for the management of obesity. The two plants namely; Kedrostis africana (L.) Cogn, Vernonia mespilifolia Less. were used singly and in combination. According to ethnobotanical studies, these two plants are regarded as wild plants and are only used for medicinal purposes. There has been a dearth of scientific reports on the two plants and to the best of our knowledge, this study is the first to investigate the nutritional, antioxidant, antimicrobial, safety and anti-obesity potentials of the two plants and their combination. This study revealed that both plants are rich in nutrients. K. africana had greater ash (16.28%), crude fat (1.12%), Ca (2505 mg/100g), Mg

(485 mg/100g) and Fe (89.95 mg/100g) while V. mespilifolia showed higher crude fibre

(29.24%), crude protein (10.75%), P (400 mg/100g), Na (570 mg/100g), Cu (1.55 mg/100g) and Mn (4.70 mg/100g). K. africana and V. mespilifolia both contributed 223.37 Kcal/kg and

237.37 Kcal/kg of energy respectively.

The polyphenolic evaluation of the acetone, aqueous and ethanol extracts of the plants revealed that the acetone extract of the combination of both plants had higher total phenol (144.59 mgGAE/g) and flavonoid (803.93 mgQE/g) contents, the ethanol extract of the combined plant also had the highest tannin contents (1.25 mgTAE/g), while the acetone extracts of K. africana had the highest proanthocyanidin content (585 mgCE/g). The antioxidant assays revealed that the ethanol extract for V. mespilifolia had higher scavenging potentials of ABTS, DPPH and

nitric oxide radicals while the aqueous extract of the combination of both had higher scavenging potential for hydrogen peroxide radicals.

Evaluation of antimicrobial potential of the extracts using the minimial inhibitory concentration

(MIC) assay against 6 bacteria (Actinomyces odontolyticus, Lactobacillus sakei,

Staphylococcus aureus, Enterobacter cloacae, Pseudomonas aeruginosa and Bacteriodes thetaiotomicron) and 4 fungi (Candida albicans, Microsporium gypsum, Penicillium chrysogenum and Trichophyton tonsurans) revealed that the plants possess antimicrobial activity. The MIC of the extracts against the tested bacterial strains ranged from 2.5 mg/mL to

5 mg/mL acetone and ethanol extracts of V. mespilifolia and the combined plants as well as the ethanol extracts of K. africana. However, no activity was observed for the acetone extract of

K. africana and aqueous extracts of V. mespilifolia and the combination of both plants. Only three fungal strains (Candida albicans, Microsporium gypsum and Penicillium chrysogenum) were susceptible to the organic extracts with an MIC 0.3125 mg/mL to 5 mg/mL while the aqueous extracts showed no activity against all the fungal strains. None of the plant extracts showed any activity against Trichophyton tonsurans.

The brine shrimp toxicity test revealed that all the three extracts of V. mespilifolia, aqueous and ethanol extracts of K. africana and aqueous and acetone extracts of the combination of both plants were toxic in relative to Meyer’s index and other indices of toxicity. The cytotoxic effect of the aqueous and ethanol extracts of V. mespilifolia, K. africana and their combination were also evaluated using HeLa cells. From this study, all the extracts tested had IC50 values were greater than 20 µg/mL which connotes that they are not toxic. According to the American

National Cancer Institute, crude plant extracts are considered cytotoxic in an in vitro assay when concentrations 20 µg/mL and below produce 50% inhibition of tumor cells, after an exposure time of 48 hours.

In vivo acute evaluation of single oral administration of 2000 and 5000/kg body weight did not produce mortality or significant behavioral changes during 14 days observation. In addition, the sub-acute administration of the aqueous extract at 200, 400 and 600 mg/kg/bwt/ day over a period of 28 days revealed no mortality or morbidity. The weekly body and organ weight of the rats were not significantly different from those of the controls and extract treated rats. The aqueous extracts at all doses did not show any significant (p > 0.05) effect on biomarkers of liver and renal damage. Haematological evaluation revealed that oral administration of aqueous extracts of K. africana, V. mespilifolia and the combination of both plants did not induce anaemia or leucocytosis in the animals. Furthermore, histopathological evaluation of the internal organs revealed no detectable inflammation at the the doses administered over a period of 28 days. These results demonstrated that the aqueous extracts of K. africana, V. mespilifolia and the combination of both plants was potentially safe for consumption orally even in chronic administration.

Enzyme based in-vitro antiobesity evaluation of the aqueous and ethanolic extracts of K. africana, V. mespilifolia and their combination revealed that the ethanol extracts of both plants and their combination exhibited moderate inhibitory activities against α-amylase, α- glucosidase and pancreatic lipase. However, the standards used for the various inhibition assays exhibited much higher inhibitory actions when compared to any of the extracts. This suggests that the mechanism by which these two plants and their combination exert anti-obesity effects are probably not by inhibition of key enzymes of carbohydrate and fat metabolism.

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Table of Contents Pages

DECLARATION ...... II DEDICATION ...... II ACKNOWLEDGEMENTS ...... III ETHICAL APPROVAL FOR THE STUDY ...... IV ABSTRACT ...... V TABLE OF CONTENTS PAGES ...... VIII LIST OF FIGURES ...... xii LIST OF TABLES ...... xv INTRODUCTION ...... 1 Plants have anti-obesity activity ...... 3 Research Problem ...... 4 Kedrostis africana (Linnaeus) Cogn...... 5 Folkoric medicinal uses ...... 6 Vernonia mespilifolia Less...... 6 Folkoric medicinal uses ...... 7 Plant combinations in traditional medicine ...... 8 Choice of solvents for extraction...... 8 Aims and Objectives ...... 8 The specific objectives of this project are: ...... 9 The structure of this thesis ...... 9 REFERENCES ...... 11 LITERATURE REVIEW ...... 14 INTRODUCTION ...... 16 Prevalence of obesity and its occurrence in South Africa ...... 17 Measurement of overweight and obesity ...... 18 Risk factors for obesity ...... 19 Obesity and its co-morbidities ...... 22 Management of obesity ...... 26 Non-Pharmacological approach ...... 26 Pharmacotherapeutic approach ...... 27 Surgical approach ...... 28 Herbal management of obesity ...... 28

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Mechanism of action of medicinal plants with anti-obesity properties ...... 29 REFERENCES ...... 33 NUTRITIONAL EVALUATION OF KEDROSTIS AFRICANA (L.) COGN: AN EDIBLE WILD PLANT OF SOUTH AFRICA ...... 50 ABSTRACT ...... 52 INTRODUCTION ...... 52 MATERIALS AND METHODS ...... 53 RESULT ...... 54 DISCUSSION ...... 55 REFERENCES ...... 56 ESSENTIAL OIL COMPOSITION, NUTRIENT AND ANTINUTRIENT ANALYSIS OF VERNONIA MESPILIFOLIA LESS...... 59 ABSTRACT ...... 61 INTRODUCTION ...... 62 MATERIALS AND METHODS ...... 62 RESULTS AND DISCUSSION ...... 63 CONCLUSION ...... 66 REFERENCES ...... 66 PHYTOCHEMICAL CONTENTS AND ANTIOXIDANT ACTIVITIES OF KEDROSTIS AFRICANA, VERNONIA MESPILIFOLIA AND THEIR COMBINATION ...... 69 INTRODUCTION ...... 71 METHODOLOGY ...... 72 RESULTS AND DISCUSSION ...... 79 CONCLUSION ...... 91 REFERENCES ...... 92 EVALUATION OF THE ANTI-MICROBIAL ACTIVITIES OF KEDROSTIS AFRICANA, VERNONIA MESPILIFOLIA AND THEIR COMBINATION ON MICROBES ASSOCIATED WITH OBESITY ...... 99 INTRODUCTION ...... 101 MATERIALS AND METHODS ...... 102 RESULTS ...... 105 DISCUSSION ...... 108 CONCLUSION ...... 110 REFERENCES ...... 111 TOXICITY EVALUATION OF VERNONIA MESPILIFOLIA LESS (A SOUTH AFRICA MEDICINAL PLANT) USING BRINE SHRIMP ...... 116

Abstract ...... 118 Introduction ...... 119 Materials and Methods ...... 119 Results and Discussion ...... 120 Conclusion ...... 124 References ...... 124 TOXICITY ASSESSMENT OF KEDROSTIS AFRICANA: A MEDICINAL PLANT USED IN THE MANAGEMENT OF OBESITY IN SOUTH AFRICA USING BRINE SHRIMP ASSAY ...... 126 ABSTRACT ...... 128 INTRODUCTION ...... 129 MATERIALS AND METHODS ...... 130 RESULT ...... 132 DISCUSSION ...... 139 CONCLUSION ...... 141 REFERENCES ...... 142 TOXICITY EVALUATION OF EXTRACTS FROM THE COMBINATION OF KEDROSTIS AFRICANA AND VERNONIA MESPILIFOLIA USING BRINE SHRIMP MODEL ...... 145 INTRODUCTION ...... 147 MATERIALS AND METHODS ...... 147 RESULTS AND DISCUSSION ...... 149 CONCLUSION ...... 157 REFERENCES ...... 158 CYTOTOXICITY EVALUATION OF KEDROSTIS AFRICANA, VERNONIA MESPILIFOLIA AND THE COMBINATION OF BOTH PLANTS USING HELA CELL LINE ...... 161 INTRODUCTION ...... 163 MATERIALS AND METHODS ...... 163 RESULTS AND DISCUSSION ...... 165 CONCLUSION ...... 171 REFERENCES ...... 172 IN VIVO TOXICOLOGICAL EVALUATION OF AQUEOUS EXTRACTS OF KEDROSTIS AFRICANA, VERNONIA MESPILIFOLIA AND THE COMBINATION OF BOTH PLANTS USING WISTAR RAT MODEL ...... 175 INTRODUCTION ...... 177 MATERIALS AND METHOD ...... 178

RESULTS AND DISCUSSION ...... 182 CONCLUSION ...... 201 REFERENCES ...... 202 EVALUATION OF THE ANTI-OBESITY POTENTIAL OF KEDROSTIS AFRICANA, VERNONIA MESPILIFOLIA AND THE COMBINATION OF BOTH PLANTS USING ENZYME-BASED IN-VITRO ASSAYS ...... 205 INTRODUCTION ...... 207 MATERIALS AND METHODS ...... 208 RESULTS AND DISCUSSION ...... 212 CONCLUSION ...... 218 REFERENCES ...... 219 GENERAL DISCUSSION AND CONCLUSION ...... 223 DISCUSSION ...... 224 CONCLUSIONS ...... 228 REFERENCES ...... 230 APPENDIX ...... 232

LIST OF FIGURES

Figure 1. 1: Bulbs of Kedrostis africana ...... 6 Figure 1. 2: Whole plant of Vernonia mespilifolia ...... 7

Figure 2. 1: Obesity-induced macrophage infiltration into adipose tissue causes insulin resistance. (A) In adipose tissue in a lean state, most resident macrophages are M2 macrophages that contribute to insulin sensitivity by secreting IL-10. (B) Hyperphagia and lack of exercise cause hypertrophy of adipocytes, which induces MCP-1 secretion to the circulation, leading to the recruitment of circulating monocytes to adipose tissues. (Source: Tateya et al. 2013)...... 24

Figure 5. 1: DPPH radical scavenging activity of different extracts from A) V. mespilifolia, B) K. africana, and C) combination of both plants at different concentrations. Each value represents mean ± SD (n = 3)...... 83 Figure 5. 2: ABTS radical scavenging activity of different extracts from A) V. mespilifolia, B) K. africana, and C) combination of both plants at different concentrations. Each value represents mean ± SD (n = 3) ...... 85 Figure 5. 3: Nitric oxide radical scavenging activity of different extracts from a) V. mespilifolia, b) K. africana, and c) combination of both plants at different concentrations. Each value represents mean ± SD (n = 3)...... 87 Figure 5. 4: Inhibition of hydrogen peroxide activity of different extracts from a) V. mespilifolia, b) K. africana, and c) combination of both plants at different concentrations. Each value represents mean ± SD (n = 3)...... 89

Figure 8. 1: Percentage hatching success of A. salina cysts incubated in different solvent extracts of K.africana and controls. The values are means of five concentrations for each plant extract/control ± SD of three replicates. Bars with different letters are significantly different (p < 0.05)...... 133 Figure 8. 2: Percentage hatching success of A. salina cysts incubated in different solvent concentrations of K.africana and control. The values are means of the replicates (at different hours) for the concentrations for each plant extract/control ± SD of three replicates. Set of bars with different letters is significantly different (p < 0.05)...... 134 Figure 8. 3: Percentage hatching success of A. salina cysts incubated at different durations in K.africana extracts/controls. The values are means of replicates (of all the concentrations) for each plant extract/control ± SD of three replicates. Set of bars with different letters are significantly different (p < 0.05)...... 135 Figure 8. 4: Percentage mortality of A. salina nauplii incubated in different solvent extracts of K. africana and controls. Means are values of five concentrations for each plant fraction/control ± SD of three replicates. Bars with different letters are significantly different (p < 0.05)...... 136 Figure 8. 5: Percentage mortality of A. salina cysts incubated in different concentrations of the extracts of K.africana and control. The values are means of the replicates (at different hours) for the concentrations for each plant extract/control ± SD of three replicates. Set of bars with different letters are significantly different (p < 0.05)...... 137 Figure 8. 6: Percentage mortality of A. salina cysts incubated in different time durations in extracts of K.africana /controls. The values are means of replicates (of all the concentrations) for each plant extract/control ± SD. Set of bars with different letters are significantly different (p < 0.05)...... 138

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Figure 9. 1: Percentage hatching success of A. salina cysts incubated in different solvent extracts of combination of K. africana and V. mespilifolia and controls. The values are means of five concentrations for each plant extract/control ± SD of three replicates. Bars with different letters are significantly different (p < 0.05)...... 150 Figure 9. 2: Percentage hatching success of A. salina cysts incubated in different concentrations of the extracts of combination of K. africana and V. mespilifolia and control. The values are means of the replicates (at different hours) for the concentrations for each plant extract/control ± SD of three replicates. Set of bars with different letters is significantly different (p < 0.05)...... 151 Figure 9. 3: Percentage hatching success of A. salina cysts incubated at different durations in extracts of combination of K. africana and V. mespilifolia/controls. The values are means of replicates (of all the concentrations) for each plant extract/control ± SD of three replicates. Set of bars with different letters are significantly different (p < 0.05)...... 152 Figure 9. 4: Percentage mortality of A. salina nauplii incubated in different solvent extracts of combination of K. africana and V. mespilifolia plant extracts and controls. Means are values of five concentrations for each plant fraction/control ± SD of three replicates. Bars with different letters are significantly different (p < 0.05)...... 153 Figure 9. 5: Percentage mortality of A. salina cysts incubated in different concentrations of the extracts of combination of K. africana and V. mespilifolia and control. The values are means of the replicates (at different hours) for the concentrations for each plant extract/control ± SD of three replicates. Set of bars with different letters is significantly different (p < 0.05)...... 154 Figure 9. 6: Percentage mortality of A. salina cysts incubated in different time durations in the plant extracts/controls. The values are means of replicates (of all the concentrations) for each plant extract/control ± SD. Set of bars with different letters are significantly different (p < 0.05)...... 156

Figure 10. 1: Hoechst (blue) and Propidium iodide (red) staining of HeLa cells following 48 hours exposure to aqueous and ethanol extracts V. mespilifolia at 200 μg/mL and the positive control (Melphalan; 100 μg/mL). Blue staining indicates live cells; red staining indicates dead cells. Where a= untreated control group; b= positive control (Melphalan; 100 μg/mL); c= aqueous extract at 100 μg/mL and d=ethanol extract at 100 μg/mL...... 166 Figure 10. 2: Hoechst (blue) and Propidium iodide (red) staining of HeLa cells following 48 hours exposure to aqueous and ethanol extracts K. africana at 200 μg/mL and the positive control (Melphalan; 100 μg/mL). Blue staining indicates live cells; red staining indicates dead cells. Where a= untreated control group; b= positive control (Melphalan; 100 μg/mL); c= aqueous extract at 100 μg/mL and d=ethanol extract at 100 μg/mL...... 167 Figure 10. 3: Hoechst (blue) and Propidium iodide (red) staining of HeLa cells following 48 hours exposure to aqueous and ethanol extracts of the combination of both plants at 200 μg/mL respectively and the positive control (Melphalan; 100 μg/mL). Blue staining indicates live cells; red staining indicates dead cells. Where a= untreated control group; b= positive control (Melphalan; 100 μg/mL); c= aqueous extract at 100 μg/mL and d=ethanol extract at 100 μg/mL...... 168 Figure 10. 4: HOE/PI cytotoxicity (expressed as % of control ± standard deviation; n = 3) of a) aqueous extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. b) ethanol extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. Values are mean ± SD (n = 3). Mean separation by LSD (p < 0.05). Set of bars (the same concentration) with different alphabets are different...... 170

Figure 12. 1: Inhibition of pancreatic lipase activity of a) aqueous extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. b) ethanol extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. Values are mean ± SD (n = 3). Mean separation by LSD (p < 0.05). Set of bars (the same concentration) with different alphabets are different. @Lower than orlistat (positive control)...... 214

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Figure 12. 2: Inhibition of alpha amylase activity of a) aqueous extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. b) ethanol extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. Values are mean ± SD (n = 3). Mean separation by LSD (p < 0.05). Set of bars (the same concentration) with different alphabets are different. #Lower than acarbose (positive control)...... 216 Figure 12. 3: Inhibition of alpha glucosidase activity of a) aqueous extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. b) ethanol extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. Values are mean ± SD (n = 3). Mean separation by LSD (p < 0.05). Set of bars (the same concentration) with different alphabets are different. $Lower than EGCG (positive control)...... 218

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LIST OF TABLES Table 2. 1. WHO ranges for BMI status ...... 19

Table 5. 1. Polyphenolic contents of various solvent extracts of V. mespilifolia, K. africana and their combination...... 81 Table 5. 2. IC50 values of the solvent of V. mespilifolia, K. africana and their combination and standard drugs ...... 90

Table 6. 1. Minimum Inhibitory Concentrations (MICs) of the different solvent extracts of V. mespilifolia, K. africana, their combination and ciprofloxacin on selected gram-negative and gram- positive bacterial strain associated with obesity...... 106 Table 6. 2. Minimum Inhibitory Concentration (MICs) of the different solvent extracts of V. mespilifolia, K. africana and their combination and nystatin on selected fungal isolates associated with obesity ...... 107

Table 8. 1. Lethal dose concentration (LD50) of acetone, ethanol and aqueous extracts of K. africana against Brine Shrimp ...... 138

Table 9. 1. Lethal dose concentration (LC50) of acetone, ethanol and aqueous extracts of combination of both V. mespilifolia and K. africana against Brine Shrimp ...... 157

Table 10. 1. IC50 values (µg/ml) of plant extract treatment of HeLa cells after 48 hours ...... 171

Table 11. 1. Effect of aqueous extracts of a) V. mespilifolia, b) K. africana and c) combination of both plants at different concentrations on food and water intake consumption by rats during the 28-days treatment...... 183 Table 11. 2. Body weights(g) of female and male rats following 28-days subacute oral administration of different doses of a) V. mespilifolia, b) K. africana, and c) combination of both plants at different dose...... 185 Table 11. 3. Relative organ weights (per 100g body weight) of rats in subacute toxicity of aqueous extracts of a) V. mespilifolia, b) K. africana and c) combination of both plants...... 188 Table 11. 4. Haematological values of rats in subacute toxicity of V. mespilifolia ...... 191 Table 11. 5. Haematological values of rats in subacute toxicity of K. africana ...... 192 Table 11. 6. Haematological values of rats in subacute toxicity of the combination of both plants .. 193 Table 11. 7. Effect of daily administration of whole plants of V. mespilifolia extract for 28 days on biochemical profiles on both sexes of control and treated rats in sub-acute toxicity study...... 196 Table 11. 8. Effect of daily administration of tubers of K.africana extract for 28 days on biochemical profiles on both sexes of control and treated rats in sub-acute toxicity study...... 197 Table 11. 9. Effect of daily administration of the combination of both plants extract for 28 days on biochemical profiles on both sexes of control and treated rats in sub-acute toxicity study...... 198

CHAPTER ONE

Introduction

CHAPTER ONE

INTRODUCTION

Contents Pages

Plants have anti-obesity activity ...... 3

Research Problem ...... 4

Kedrostis africana (Linnaeus) Cogn...... 5

Folkoric medicinal uses ...... 6

Vernonia mespilifolia Less...... 6

Folkoric medicinal uses ...... 7

Plant combinations in traditional medicine ...... 8

Choice of solvents for extraction...... 8

Aims and Objectives ...... 8

The specific objectives of this project are: ...... 9

The structure of this thesis ...... 9

References ...... 11

INTRODUCTION

Obesity is a common health disorder of lipid and carbohydrate metabolism which results from excessive fat accumulation in the adipose tissue, liver, skeletal muscle etc. (WHO, 2011). The condition is characterized by increase in adipose cell size due to large amount of fat deposited in the cytoplasm of adipocytes (Delvin et al. 2000). Obesity occur as a result of imbalance between energy intake and energy expenditure during an extended period of time. The imbalance could be attributed to excess energy intake relative to daily energy expenditure, or as low energy expenditure relative to daily energy intake, brought about by the impairment of lipid metabolic processes such as lipogenesis and lipolysis (Pagliassotti et al. 1997). This metabolic imbalance often leads to multiple pathologies such as heart failure, Type II diabetes and cancers. In South Africa, like other developing and developed countries, urbanization and socio-economic development are accompanied by a change in diet towards more high energy dense foods (more meat, fat, salt and sugary foods) as well as a reduction in physical activity

(mechanized transport) resulting in increased storage of the excess calories as fat in adipose tissue (Jorge et al. 2014). This situation is further aggravated by the antiquated African belief that obesity especially among women is a positive indicator of the good health, wealth, beauty, wealth and good reproductive health (Micklesfield et al. 2013).

Plants have anti-obesity activity Plants have been used as traditional natural medicines for healing many diseases. Medicinal plants have been investigated and reported to be useful in the treatment of obesity, diabetes and other chronic diseases. Herbal supplements and diet-based therapies for weight loss are among the most common complementary and alternative medicine (CAM) options. A large array of natural products, including crude extracts and isolated compounds from plants can be used to induce weight loss and prevent diet-induced obesity. In addition, literature has revealed that

various medicinal plants such Citrus aurantium, Nigella sativa, and Camellia sinensis are used traditionally in the management of obesity (Moro and Basile, 2007; Ahn et al. 2010; Hasani-

Ranjbar et al. 2013). In the Eastern Cape Province of South Africa in particular, several plants traditionally used for the prevention, treatment and management of obesity have been reported and documented (Afolayan and Mbaebie, 2010).

Research Problem The geometric increase in the prevalence of obesity constitutes a global public health challenge, especially in developing nations where people have adopted more modernized and sedentary lifestyles as well as the consumption of diets that predispose to becoming obese. Antiobesity therapy such as adjustment in food intake, physical activity and the use of conventional drugs are ways by which obesity can be managed. Several pharmacological substances available as anti-obesity drugs have hazardous side effects (NIH, 1998; Christensen et al. 2007). Many natural products have been used traditionally in most cultures for treating obesity. However, the potential of natural products especially from medicinal plants for the treatment of obesity is still largely unexplored and can be an excellent alternative for the safe and effective development of anti-obesity drugs. The currently available drugs for the treatment of obesity are orlistat-which reduces fat absorption through inhibition of pancreatic lipase; and sibutramine which is an anorexic or an appetite suppressant. Both drugs have adverse effects including increased blood pressure, headache, dry mouth, insomnia, and constipation (Drew et al. 2007; Tziomalos et al. 2009). According to Lowes (2010), sibutramine has been withdrawn from the market since October 2010 by the FDA because of increased cardiovascular events and strokes (James et al. 2010). Currently only orlistat (Xenical), a drug which is considered to act through inhibition of pancreatic lipase enzyme, a key enzyme for the digestion of dietary triglycerides, has been approved by FDA and is available for long-term treatment of obesity

(Guerciolini, 1997; Kang and Park, 2012). 4

As a result of these potentially hazardous side effects and high cost, there is an urgent need for the exploration of natural products from herbs, spices and other medicinal plants with anti- obesity activities which could serve as alternatives to the synthetic drugs or leads for developing effective and safe anti-obesity drugs.

Rationale and justification for study

The use of medicinal plants for the management of obesity have been well documented as promising sources for the development of cheaper, cost-effective anti-obesity agents with fewer side effects that will prove more affordable for patients. However, despite their antiobesity potential, their efficacy and safety remains to be scientifically proven. Kedrostis africana, Vernonia mespilifolia, and the combination of both plants are among such plants used traditionally to manage obesity in the Eastern Cape Province of South Africa. Their ethnobotanical uses against obesity are yet to be validated in any known study. Selection of the two plants was based on their inclusion on the list of plants documented for the treatment of obesity in the Eastern Cape (Afolayan and Mbaebie, 2010), and the fact that these potentials have not been validated in any known scientific study.

Kedrostis africana (Linnaeus) Cogn. Kedrostis africana (Linnaeus) Cogn. is a monoecious caudiciform plant, commonly known as

Baboon's Cucumber or locally called Uthuvishe and Uthuvana in Xhosa language. It belongs to the family Cucurbitaceae. It is an herbaceous climbing or creeping vines growing rapidly from the swollen base, resembling an English ivy with a tuber. The shoots emerge from a massive underground tuberous rootstock (Figure 1.1). This tuber is a water-storage organ that is very resistant to drought (Eggli, 2002). The specie is native to Namibia and South Africa

(Eastern Cape, Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Northern Cape,

North West and Western Cape).

Figure 1. 1: Bulbs of Kedrostis africana

Folkoric medicinal uses According to van Wyk, (2008), Kedrostis africana tuber is used in traditional medicine as an emetic, purgative, diuretic, dropsy and to treat syphilis. Also, decoction of the crushed fresh bulb is taken twice daily for the management of obesity (Afolayan and Mbaebie, 2010; George and Nimmi, 2011).

Vernonia mespilifolia Less. Vernonia mespilifolia belongs to the family Asteraceae. It is a species that can thrive across a wide range of diverse and climatic conditions including tropical forest; marshes wet areas, dry plains, tropical savannahs, desert xeric or dry sites and even frosty regions of eastern North

America (Keeley and Jones, 1979). The genus is morphologically made up of annuals, herbaceous perennials, lianas, shrubs, and trees.

The Vernonia family possesses simple leaves with alternate or opposite leaf arrangement

(Olorode, 1984) and is known for having several species with food, medicinal and industrial 6

uses. Among the species found in South Africa is Vernonia mespilifolia (Dold and Cocks,

1999), which is also known as Iron weed (English) and Uhlunguhlungu (Xhosa). It is a half- climbing and sparsely branched perennial shrub growing up to 1.3–1.5 m in height (Figure 3.2).

The membranous leaf blade is 2-3 inches broad and 2 inches long, narrowing gradually to slender stalk.

Figure 1. 2: Whole plant of Vernonia mespilifolia

Folkoric medicinal uses According to Afolayan and Mbaebie (2010) V. mespilifolia is used for the treatment of weight loss, hypertension and as a diuretic agent. It has also been reported that this plant is combined with K. africana for the management of obesity in the Eastern Cape (Afolayan and Mbaebie,

2010). Also, the boiled stem part of the plant is used for treating heartwater disease in goats

(Dold and Cocks, 2001). These plants were authenticated by Mr. Tony Dold of Selmar

Schonland Herbarium, Rhodes University, South Africa, and a voucher specimen (Unuofin

Med, 2015/1&2) was prepared and deposited at the Giffen Herbarium, University of Fort Hare.

Plant combinations in traditional medicine The concept of combining different plant species for the management or treatment of diseases have been practiced since ancient times. In most traditional medicine systems of the world including African traditional medicine, different plant species are often combined for better efficacy (Van Vuuren and Viljoen, 2011). However, studies dedicated to validating the use of

South African plant species in combination for the management of obesity are limited. The information on the use of the selected plants was, however, obtained from the readily available literature only. Thus, there is still a need for further scientific evidence on plant combinations used by the local ethnic people. Investigating the interactive efficacy of the ethno-directed plant combinations may serve as the foundation for novel chemotherapeutic agents useful in the management of obesity.

Choice of solvents for extraction

In this study, acetone, aqueous and ethanol extracts were used. The ethanol and aqueous extracts were selected on the basis of how traditional practitioners prepare herbal medicine (s) from Kedrostis africana, Vernonia mespilifolia, and the mixture of both plants as either infusion or decoction using water or alcohol (Afolayan and Mbaebie, 2010). Acetone was also selected as an extractant because of its ability to extract both hydrophilic and lipophilic components from plants as well as volatility and low toxicity for use in microbial bioassays

(Eloff, 1998).

Aims and Objectives The overall aim of this study was to evaluate, and validate the pharmacological potential of

Kedrostis africana, Vernonia mespilifolia, and the combination of both plants in the management of obesity.

The specific objectives of this project are:  To evaluate the nutritive and mineral content of K. africana and V. mespilifolia.

 To quantitatively evaluate the phytochemical constituents and to screen for free radical

scavenging/antioxidant potential of K. africana and V. mespilifolia and the combination

of both plants.

 To evaluate the antimicrobial activities of K. africana and V. mespilifolia and the

combination of both plants on gut microbes associated with obesity

 To evaluate the toxicity threshold of K. africana and V. mespilifolia and the

combination of both plants using brine shrimp, HeLa cell lines and Wistar rat models.

 To determine the anti-obesity potentials of K. africana and V. mespilifolia and the

combination of both plants using in vitro enzymatic assays.

The structure of this thesis This thesis is composed of 13 chapters that have been published, accepted or which are under review in various peer-review and accredited journals. The general introduction is in Chapter

1. Chapter 2 presents the literature review on obesity, its prevalence and various ways of management. In Chapter 3 the nutritional evaluation of Kedrostis africana was determined.

Chapter 4 reports the essential oil composition, nutrient and anti-nutrient analysis of Vernonia mespilifolia was determined. Both plants are regarded as wild plants and as such are not consumed by the people of the Eastern Cape. Hence, these two chapters gave an insight to their nutritive potentials. Chapter 5 is an evaluation of the quantitative phytochemical and antioxidant activities of Vernonia mespilifolia, Kedrostis africana and the combination of both plants. The free radical scavenging potential of extracts is dependent on the quantity of the phytochemicals present in such extract. The different solvents were employed to ascertain what

solvent best extracts certain phytochemicals in the plant. Chapter 6 described the antifungal and antibacterial activities of Vernonia mespilifolia, Kedrostis africana and the combination of both plants the plant against a variety of selected microbes associated with obesity. Chapter 7 accounts for the toxicity evaluation of Vernonia mespilifolia using brine model. Chapter 8 is composed of the report of the toxicity assessment of Kedrostis africana using brine shrimp model. Toxicity evaluation of extracts from the combination of Vernonia mespilifolia and

Kedrostis africana using brine shrimp model is reported in Chapter 9. Chapter 10 accounts for the cytotoxicity evaluation of Kedrostis africana, Vernonia mespilifolia and the combination of both plants using HeLa cell line. In vivo toxicological evaluation of aqueous extracts of

Kedrostis africana, Vernonia mespilifolia and the combination of both plants using Wistar rat model is reported in Chapter 11. Chapter 12 presents the results of enzyme based in vitro inhibitory activities of V. mespilifolia, K. africana and the combination of both plants against

α-amylase, α-glucosidase and pancreatic lipase. The general discussion, conclusions and recommendations emanating from the entire study are presented in Chapter 13.

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obesity remedies in Nkonkobe Municipality of South Africa. Pharmacognosy

Magazine 2 (11), 368-373.

Ahn, J., Lee, H., Kim, S., Ha, T., 2010. Curcumin-induced suppression of adipogenic

differentiation is accompanied by activation of Wnt/β-catenin

signalling. American Journal of Physiology: Cell Physiology, 298, C1510–

C1516,

Christensen, R., Kristensen, P.K., Bartels, E.M., Bliddal, H., Astrup, A., 2007. Efficacy and

safety of the weight-loss drug rimonabant: a meta-analysis of randomised trials.

Lancet 370, 1706–1713.

Devlin, M.J., Yanovski, S.Z., Wilson, G.T., 2000. Obesity: what mental health professionals

need to know. American Journal of Psychiatry 157, 854-866.

Dold, A.P., Cocks, M.L., 1999. Preliminary list of plant names from Eastern Cape, South

Africa. Bothalia (29)2, 267–292.

Drew, B.S., Dixon, A.F., Dixon, J.B., 2007. Obesity management: update on orlistat. Vascular

Health and Risk Management 3, 817-821.

Eggli Urs 2002. Illustrated Handbook of Succulent Plants: Dicotyledons” Springer Science &

Business Media.

Eloff, J.N., 1998. Which extractant should be used for the screening and isolation of

antimicrobial components from plants? Journal of Ethnopharmacology 60(1), 1–

George, P., Nimmi, O.S., 2011. Cent percent safe centum plants for antiobesity. International

Journal of Innovative Technology and Creative Engineering 1(3), 1-19.

Guerciolini, R., 1997. Mode of action of orlistat. International Journal of Obesity and Related

Metabolic Disorders 21 (3), S12–S23.

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medicinal plants - an update. Journal of Diabetes and Metabolic Disorders 12, 28.

James, W.P., Caterson, I.D., Coutinho, W., Finer, N., Van Gaal, L.F., Maggioni, A.P., Torp-

Pedersen, C., Sharma, A.M., Shepherd. G.M., Rode, R.A., Renz, C.L., 2010.

Effect of sibutramine on cardiovascular outcomes in overweight and obese

subjects. The New England Journal of Medicine 363, 905-917.

Jorge, C., Zoltan, P., Alain, G., 2014. Comprendre l’obésité en Afrique: Poids du

développement et des représentations. Revue médicale suisse 2014, 712–716.

Kang, J.G., and Park, C.Y., 2012. Anti-Obesity Drugs: A Review about Their Effects and

Safety. Diabetes Metabolism Journal 36 (1), 13–25.

Keeley, S.C., Jones, SB., 1979. Distribution of pollen types in Vernonia (Vernonieae:

Compositae). Systematic Botany 4, 195–202.

Lowes, R., 2010. Abbott Withdraws Sibutramine From Market - Medscape - Oct 08, 2010.

Micklesfield, L.K., Lambert, E.V., Hume, D.J, Chantler, S., Pienaar, P.R., Dickie, K.,

Goedecke, J.H., Puoane, T., 2013. Socio-cultural, environmental and behavioural

determinants of obesity in black South African women. Cardiovascular Journal of

Africa 24 (9), 369–375.

Moro, C.O., and Basile, G., 2007. Obesity and medicinal plants. Fitoterapia 71 (1), S73–S82.

National Institutes of Health 1998. Clinical guidelines on the identification, evaluation, and

treatment of overweight and obesity in adults: the evidence report. Obesity

Research. 6(Suppl 2), 51S–209S.

Olorode O. 1984.Taxonomy of West Africa Flowering Plants. Longman, London.

Pagliassotti, M.J., Gayles, E.C., Hill, J.O., 1997. Fat and energy balance. Annals of the New

York Academy of Sciences 827, 431-448.

Tziomalos, K., Krassas, G.E., Tzotzas, T., 2009. The use of sibutramine in the management of

obesity and related disorders: an update. Vascular Health and Risk Management

5, 441-452.

Van Vuuren, S.F., Viljoen, A.M., 2011. Plant-based antimicrobial studies-methods and

approaches to study the interaction between natural products. Planta Medica 77,

1168- 1182. vanWyk. A.B.E., 2008. Review of Khoi-San and Cape Dutch medical ethnobotany. Journal of

Ethnopharmacology 119, 331–341.

WHO, 2011. Overweight and Obesity fact sheet.

CHAPTER TWO

Literature Review

CHAPTER TWO

LITERATURE REVIEW

Contents Pages

Introduction ...... 16

Prevalence of obesity and its occurrence in South Africa ...... 17

Measurement of overweight and obesity ...... 18

Risk factors for obesity ...... 19

Obesity and its co-morbidities ...... 22

Management of obesity ...... 26

Non-Pharmacological approach ...... 26

Pharmacotherapeutic approach ...... 27

Surgical approach ...... 28

Herbal management of obesity ...... 28

Mechanism of action of medicinal plants with anti-obesity properties ...... 29

References ...... 33

INTRODUCTION Obesity is increasing progressively worldwide and has become a fearsome threat to large communities. The global prevalence of obesity has more than doubled between 1980 and 2014

(WHO, 2015). According to WHO about 1.9 billion adults (18 years and older) worldwide are overweight and about 600 million are clinically obese, about 13% of the world’s adult population (11% of men and 15% of women) are obese (WHO, 2014). Correspondingly, the prevalence of childhood overweight is increasing worldwide, especially in Africa and Asia.

Between the year 2000 and 2013, the prevalence of overweight in children aged under 5 years, progressively increased from 11% - 19% in some countries in southern Africa and from 3% -

7% in South-East Asia (WHO, 2014). Moreover, the prevalence of overweight and obesity in

Africa is increasing alarmingly and it is estimated that 25% to 60% of urban women are overweight (WHO, 2013).

Obesity is a risk factor for heart disease, diabetes, stroke, and cancer. It is a leading cause of blindness, renal failure, and limb amputation for non-traumatic reasons. The total cost of this disease is approaching $130 billion per year in the United States alone and accounts for 14% of all cancer deaths in men and 20% in women (Aggarwal, 2010). Although caloric intake is one of the major contributors to obesity, it is also generally believed that highly processed, packaged, and refined foods loaded with sugar and hydrogenated oils are promoters of obesity.

Knowledge of traditional medicine has helped in the identification foods, food supplements, herbs, and spices believed to exhibit anti-obesity effects. Some of these can be considered as components of the complementary and alternative medicine (CAM) pharmacopoeia. Most recent studies have focused on the potential role of plants for the treatment of obesity and its metabolic disorders, through their positive effects on lipid and glucose metabolism and anti- inflammatory activity. Development of inhibitors of nutrient digestion and absorption, which

reduce energy intake through gastrointestinal mechanism without altering any central mechanisms, is one of the most important methods in the treatment of obesity. At present, the potential of medicinal plants for the treatment of obesity is still largely unexplored and scientifically validated. This could be an excellent alternative strategy for the development of safe and eﬀective anti-obesity drugs (Rani et al. 2012).

Prevalence of obesity and its occurrence in South Africa The prevalence of overweight and obesity is increasing, with obesity being estimated to be the second leading cause of mortality and morbidity, causing about 2.6 million deaths worldwide

(Sengwayo et al. 2012). In Africa, the prevalence of obesity is increasing alarmingly and it is estimated that 25% to 60% of urban women are overweight. South Africa, as a developing nation, is also struck by the burden of overweight and obesity and according to the World

Health Organisation statistics, more than 29% of South African men and 56% of South African women are overweight or obese (Camacho, 2011).

The Southern African region is mainly disturbed by an increasing trend in Body Mass Index

(BMI). South Africa a middle income country is displaying worrisome pictures in relation to its rapid epidemiologic transition. In 2008, the average BMI at population level was estimated at 26.9 kg/m2 among males against a world average of 23.8 kg/m2 and 29.5 kg/m2 among females against a world average of 24.1 kg/m2. Furthermore, the rate of growth between 2000 and 2008 was calculated to be 2.9 kg/m2 and 1.6 kg/m2 per decade for males and females respectively. This astonishing increase is compared to the preceding period between 1980 and

2000, where the average rates of growth were 0.7 kg/m2 and 0.65 kg/m2 per decade, respectively among males and females (Finucane et al. 2011).

Of late, this figure has risen progressively such that obesity in South Africa is highly prevalent in all sectors. According to 2015 reports from the Heart and Stroke Foundation, South Africa

has the highest overweight and obesity rate in sub-Saharan Africa, with up to 70% of women and a third of men being classified as overweight or obese (www.heartfoundation.co.za).

However, obesity is no longer just an adult problem, as 1 in 4 girls and 1 in 5 boys between the ages of 2 – 14 years are overweight or obese (www.heartfoundation.co.za). Unfortunately, obesity and its burden of diseases (type 2 diabetes, heart disease, stroke, hypertension, joint pain and certain cancers) negatively affect the life of many South Africans, promote poverty and contribute to the increasing cost of health care (Bradshaw et al. 2000). It is also one of the major contributors to morbid conditions like type-2 diabetes, hypertension, cardiovascular disorders, and non-alcoholic fatty liver metabolic syndrome (Lee et al. 2003).

Measurement of overweight and obesity The most common method for measuring obesity is the Body Mass Index (BMI). This is achieved by dividing a person’s weight measurement (in kilograms) by the square of their height (in metres). BMI is the best way to measure the prevalence of obesity at the population level. Height and weight data is used to calculate BMI; while waist circumference is used to assess central obesity in adults.

BMI is calculated as BMI= Weight (kg)/ Height (m2)

According to WHO, to achieve optimum health, the median body mass index for an adult population should be in the range of 21 - 23 kg/m2, while the goal for individuals should be to maintain a body mass index in the range 18.5 - 24.9 kg/m2. There is increased risk of comorbidities for body mass index 25.0 - 29.9 kg/m2, and moderate to severe risk of comorbidities for body mass index greater than 30. According to WHO(1998), BMI for various status range for 18.5 kg/m2 from underweight to 40 kg/m2 in the morbidly obese (Table 2.1).

Table 2. 1. WHO ranges for BMI status Definition BMI range (kg/m2)

Underweight ≤ 18.5

Normal 18.5 ≤ 25

Overweight 25 ≤ 30

Obese 30 ≤ 40

Obese I 30 ≤ 35

Obese II 35 ≤ 40

Morbidly obese ≥ 40

Source: World Health Organization (1998)

Risk factors for obesity Obesity is considered a multifactorial disease that originates from a combination and an interaction of factors such as insufficient sleep, endocrine disruptors, decreased variability in ambient temperature, decreased rates of smoking (as smoking suppresses appetite) (Audrain

Mc Govern and Benowitz, 2011) and increased use of medications that can cause weight gain such as atypical antipsychotics (Bak et al. 2014). Other factors include proportional increase in ethnic and age groups that tend to be heavier, pregnancy at a later age (which may cause susceptibility to obesity in children) (Chalk, 2004) and epigenetic risk factors passed on generationally (Ness-Abramof and Apovian, 2006).

Diet: The western diet provides a wide range of opportunities to consume energy-dense food and drink products, which inadvertently leads to what has been described as “passive over- consumption”, where the individual has no way of recognizing that he or she consuming in excess a particularly energy-dense food or drink (Bell and Roll, 2001). Given this perspective, it is not surprising that sweetened beverages (Mattes et al. 2001) and “fast foods” (Pereira et

al. 2005) emerge as specific risk factors for obesity. In addition, large portion sizes of energy- dense foods increase the risk of excessive consumption (Ledikwe et al. 2005), whereas the frequency of eating itself has not been shown to contribute specifically to weight change, when the type of food is the same. Therefore, higher intakes of fruit and vegetables are linked to lower weight gains (He et al. 2004), while a high intake of meat (together with its associated fat) is linked to a greater risk of weight gain (Schulz et al. 2002).

According to Mitchell et al. (2011), foods high in sugar and energy dense contribute to overeating and are currently the cheapest and most accessible. Thus, to get the most calories for the least money is to eat a diet that is high in fat and sugar.

Lifestyle and physical activity: A sedentary lifestyle is one with little or no physical activity and a person living a sedentary lifestyle is often sitting or lying down, while reading, socializing, watching television, playing video games, or using a mobile phone/computer for much of the day. A sedentary lifestyle is one of the major contributors to obesity.

Physical activity can be broadly divided into exercise and non-exercise activities. Non-exercise activities are difficult to measure and includes employment related work and the activity of daily living (Hill and Wyatt, 2005). A sedentary lifestyle could make an individual more susceptible to high energy imbalance when combined with a contemporary diet rich in energy- dense, processed foods and sugar-containing beverages (Ross and Janiszewski, 2008). It is now recognized that increased energy expenditure through physical activity has a more positive role in reducing fat stores and adjusting energy balance in the obese, especially when it is combined with modification of the diet (Hill and Wyatt, 2005).

Genetics and obesity: Genes and family history have also been attributed to the incidence of obesity (Lin et al., 2010). The interplay between genetic and environmental factors bring about

the onset of obesity. Individuals with two copies of the fat mass and obesity associated gene

(FTO gene) have been shown to have a 1.67 fold greater risk of obesity development when compared to those without risk allele (Loos and Bouchard, 2008 ). Some other cases of obesity have been attributed to single-gene mutations, e.g. melano-cortin-4 receptor (MC4R) gene, dopamine receptor D4 (DRD4), peroxisome proliferator- activated receptor y2 (PPARy2) or the leptin genes (Nothen et al. 1994; Ristow et al. 1998; Farooqi et al. 2003).

Medical and psychiatric illness: A number of antipsychotic medications have also been implicated to increase the risk of obesity. This risk is higher in patients with psychiatric disorders than in persons without psychiatric disorders (Chiles and Van Wattum, 2010). Other medical conditions such as genetic syndromes, congenital or acquired conditions like hypothyroidism, growth hormone deficiency, eating disorders (binge eating disorder and night eating syndrome) and certain medicines that are used for depression have so been found to contribute towards obesity (Haslam and James, 2005; Uguz et al. 2015).

Social factors such as age, sex, marriage status, educational level, social class (manual, non- manual), migration and monthly income are significant risk factors of predictors obesity.

Evidence suggests that obesity is socially distributed, with certain social groups at increased risk. An inverse relation between social class and body weight and risk of obesity among women, and less consistently among men in industrialized countries have been reported (Ball et al. 2003). Other sociodemographic factors such as marital status (being or getting married is associated with higher risk), migration status, employment status, family status and housing situation has also been linked with obesity (Sobal et al.1992; Ortiz-Moncada et al. 2011).

According to Ball et al. (2003) one possible explanation is that these relations are attributable to differences across social groups in levels of participation in weight-related behaviours.

Differences in eating, physical activity and other weight-related behaviours completely account 21

for differences in obesity prevalences across social groups. For instance, lower levels of recreational physical activity have been reported amongst individuals of low social class, among those who are married, and among immigrants. In addition, individuals of low social class are less likely to consume a healthy or low-fat diet (Laitinen et al. 2001; Ball et al.

2003; Saunders et al. 2012)

The effect of smoking has also been shown to influence an individual’s weight significantly.

For males and females who quit smoking, they gain an average of 4.4 kg and 5.0 kg respectively over a period of ten years (Chiolero et al. 2008).

Gut microbiota and obesity: The gut microbiota is an important factor affecting energy disposal and storage in adipocytes (Bäckhed et al. 2004; Ley et al. 2006). It is also known to be involved in modulation of host immunity, and the inflammatory status associated with obesity in mice (Cani et al. 2007a, Cani et al. 2007b). An association between viruses and obesity has been found in humans and several different animal species (Di-Baise et al. 2008).

According to Fei and Zhao (2013), the role of the gut microbiota in the pathogenesis of obesity is currently an important research area, as Gram-negative opportunistic pathogens in the gut have been implicated in the etiology of obesity (Zhang et al. 2012). Cani et al. (2007a) reported that Lipopolysaccharide (LPS) endotoxin purified from Escherichia coli induced obese and insulin-resistant phenotypes when subcutaneously infused into mice at a concentration comparable to what can be found in a mouse model of high-fat diet (HFD)-induced obesity.

Obesity and its co-morbidities Obese patients are at an increased risk for developing many medical problems, including inflammation, insulin resistance, type 2 diabetes mellitus, hypertension, dyslipidemia, cardiovascular disease, stroke, sleep apnea, gallbladder disease, gout, osteoarthritis and cancer. In addition, certain psychological problems such as depression, social stigmatization 22

and discrimination, and impaired psychosocial and physical functioning, could cause a negative impact on the quality of life of the obese. Major comorbidities associated with obesity include

Inflammation: Adipose tissue-derived proteins also known as adipokines, have been implicated in the pathogenesis of chronic inflammation in obesity. The study of adipose tissue on inflammation is demonstrated by the residence macrophages in adipose tissue (Xu et al. 2003).

The possible mechanisms fundamental to the infiltration of macrophages into adipose tissue may possibly be the chemokines by adipocytes, which would then attract resident macrophages

(Weisburg et al. 2003). Increased number of macrophages is observed in the adipose tissue of obese patients, and once activated, these macrophages secrete a myriad of cytokines, such as

TNF-α, IL-6, and IL-1. During the commencement of an inflammatory process, macrophages found in adipose tissue switch from an anti-inflammatory (M2) state to a pro-inflammatory

(M1) state (Chawla et al. 2011). These infiltrated macrophages differentiate into activated M1 macrophages, which robustly secrete proinflammatory cytokines such as TNFα, IL-6, and

MCP-1, thus contributing to low-grade inflammation in adipose tissue and a decrease of adiponectin.

Insulin resistance: Insulin is a key regulator of nearly all aspects of adipocyte biology. The adipocytes are one of the most highly insulin-responsive cells in the body. The physiological role of insulin includes the metabolism of all 3 macronutrients (carbohydrates, lipids, and proteins) as well as cellular growth. Insulin’s action on lipid metabolism is analogous to its role in glucose metabolism, i.e. promoting anabolism and inhibiting catabolism. Insulin stimulates glucose transport and triglyceride synthesis (lipogenesis), as well as inhibiting lipolysis

(Samuel et al. 2010). Precisely, insulin upregulates lipoprotein lipase (LPL) and promotes the gene expression of some intracellular lipogenic enzymes, namely acetyl-CoA carboxylase

(ACC) and fatty acid synthase (FAS). In addition, insulin inhibits adipocyte hormone sensitive lipase (HSL) through inhibition of its phosphorylation (Shen et al. 2009). Insulin action in 24

adipocytes also involves changes in gene transcription. The transcription factor ADD-

1/SREBP-1c (adipocyte determination and differentiation factor-1/sterol regulatory element- binding protein-1c) may play a critical role in the actions of insulin to regulate adipocyte gene expression (Payne et al. 2009), by inducing genes involved in lipogenesis and repressing those involved in fatty acid oxidation. Functional defects in insulin resistance could be attributed to impaired insulin signalling in some target tissues such as adipose tissue, skeletal muscle and liver (De Taeye et al. 2007). The combination of obesity and insulin resistance have been implicated in the development of type 2 diabetes mellitus (De Taeye et al. 2007) which is characterized by decrease in insulin-stimulated glucose uptake, impair hepatic glucose output

(Tesz et al. 2007) and high levels of stored lipids in skeletal muscle. In both muscle and adipocytes, the binding of insulin to its receptors causes the receptor to phosphorylates and activate tyrosine kinase and ultimately leading to the reduction in insulin receptor substrate phosphorylation which results in obesity (Singla et al. 2010). Recent studies have indicated that defective signaling from the insulin receptor is an important component of insulin resistance associated with obesity in humans (Singla et al. 2010).

Cardiovascular diseases: In human subjects, a well established relationship had been made between obesity and hypertension (DeMarco et al. 2014). The activations of sympathetic nervous system and renin- angiotensin system (RAS), insulin resistance, inflammation, dysfunction of the vascular endothelium and increased secretion of leptin are some of the mechanisms implicated in the onset of obesity and hypertension (Kotsis et al. 2010).

Studies have revealed that the impairment of both diastolic and systolic functions is peculiar among obese human subjects (Norton et al. 2009). In addition, obesity-related cardiomyopathy such as left atrial dilatation, left ventricular (LV) hypertrophy and abnormalities in left ventricular contractile among obese subjects (Norton et al. 2009; Glenn et al. 2011). Reduction

in left ventricular systolic function has also been implicated in several animal models of obesity

(Norton et al. 2009), but little variation was observed in some studies where diet-induced obese rats showed mildly reduced or systolic function (Sun et al. 2012).

Management of obesity In recent times, the available options for the treatment and management of obesity include non- pharmacological treatment, pharmacotherapy and surgical procedures, each of which has its own advantage and drawbacks.

Non-Pharmacological approach Non-pharmacological treatment consists of lifestyle modification, reduction of total caloric intake and regular aerobic exercise. This approach is preferred for many reasons such as adverse effects of anti-obesity drugs, contraindications or allergic reactions to drugs, perceptions of adverse effects of drugs, or personal preference for natural or alternative therapies. Lifestyle Modifications can be achieved by a reduction in energy intake and an increase in physical activity (Scheen, 2008). Dietary approach for reducing weight is achieved by reducing the total amount of calories consumed, and is best accomplished by a reduction in the amount of fat in the diet and calories from soft drinks (Franz et al. 2002). The components of diet currently recommended as healthy is composed of little amount of saturated and trans fats, intake of carbohydrates rich in dietary fiber, high fruit and vegetable intake, and the addition of low-fat dairy food would prevent the onset of metabolic syndrome (Feldeisen and

Tucker, 2007).

Exercise as a treatment and preventive strategy for combating obesity can not be over emphasized. A number of studies that employed the imaging techniques to quantify changes in abdominal obesity suggests a beneficial influence of physical activity on reduction of abdominal fat and visceral adipose tissue (VAT) in overweight and obese subjects (Kay and

Fiatarone, 2006). The reductions in VAT and total abdominal fat could occur in the absence of changes in body mass and waist circumference. Fats deposited in the abdomen and in nonadipose tissues such as liver (Yki-Jiirvinen, 2005) and muscle (Moro, Bajpeyi and Smith,

2008) plays a key role in the development of obesity-related health risks. These fore mentioned fat stores have emerged as promising alternative targets for obesity treatment (Janiszewsk and

Ross, 2007).

Pharmacotherapeutic approach

Clinical guidance on the use of anti-obesity drugs states that they should be an adjunct to first- line of treatments, which are, exercise and lifestyle modification (McGovern et al. 2008). There are two different types of obesity-treatment drugs which are currently available on the market-

Orlistat (Xenical), which reduces intestinal fat absorption through inhibition of pancreatic lipase (Thurairajah et al. 2005; Chaput et al. 2007; Drew et al. 2007); and Sibutramine

(Reductil), which is an anorectic, or appetite suppressant drug (Poston and Foreyt, 2004;

Tziomalos et al. 2009). Both drugs have side effects, including increased blood pressure, dry mouth, constipation, headache, and insomnia (Slovacek et al. 2008; Karamadoukis et al. 2009).

A number of anti-obesity drugs are currently undergoing clinical development, including centrally-acting drugs (e.g. Radafaxine and Oleoyl-estrone), those targeting peripheral episodic satiety signals (e.g. Rimonabant and APD356), blocking fat absorption (e.g. Cetilistat and

AOD9604), and human growth hormone fragments (Halford, 2006; Melnikova and Wages,

2006).

The British National Formulary recommends that drugs for obesity should only be considered for those with a BMI of 30 or greater if supervised diet, exercise and behaviour modification fail to achieve a realistic reduction in weight (Greenway, 1996).

Surgical approach

Bariatric surgical procedures (i.e. gastroplasty, gastric bypass) are the only procedures that provide marked and sustained weight reduction in morbidly obese patients, leading to improvements in associated metabolic disorders, especially type 2 Diabetes Mellitus, and a more favorable long-term prognosis, including a reduction in total mortality (Sjöström et al.

2007). However, considering the risk/benefit ratio of bariatric surgery, it may not yet be considered an early option in the management of the abdominally obese patient.

Herbal management of obesity

At present, because of dissatisfaction with high costs and potentially hazardous side effects of anti-obesity drugs, the potential of natural products from medicinal plants for treating obesity is under exploration, and this may be an excellent alternative strategy for developing future effective, safe anti-obesity drugs (Mayer et al. 2009; Nakayama et al. 2007; Park et al. 2005).

Although weight loss and weight control drugs are becoming extremely common, the remedies provided by the diet industry have failed in the long-term maintenance of weight loss in obese subjects (Wadden, 1993). Moreover, it has been estimated that more than 90% of the people who lose weight by dieting return to their original weight within 2–5 years (Stern et al. 1995).

Literature has shown that the majority of plants with antiobesity potential mainly belongs to the family Araliaceae, Asteraceae, Cucurbitaceae, Lamiaceae Leguminoseae, Liliaceae,

Moraceae and Rosaceae (Patra et al. 2015). A preponderance of the studies showed decrease in body weight or body weight gain in animals and humans with or without changes in body fat indicating antiobesity effects. In addition, the reduction in body weight, decrease in the levels of triglycerides, total cholesterol, and low density lipoprotein cholesterol with concurrent increase in high density lipoprotein cholesterol was noted in the animals treated with the plants

(Adeneye et al. 2010; Park et al. 2012; Mali et al. 2013; WHO, 2015). At present, because of

dissatisfaction with high costs and potentially hazardous side effects, the potential of natural products for treating obesity is under exploration, and this may be an excellent alternative strategy for developing future effective, safe anti-obesity drugs (Nakayama et al. 2007; Mayer et al. 2009)

Mechanism of action of medicinal plants with anti-obesity properties Medicnal plants with anti-obesity properties haved been classified based on their mechanism of action as (1) Peripherally acting and (2) Centrally acting.

(1) Peripherally acting: Peripherally acting medicinal plants act by exerting their effects

through the reduction of calorie absorbed in the gastrointestinal system (Birari and

Bhutani, 2007). These effects are carried out by

(a) Lipase Inhibition: Dietary fats are not immediately taken up by the intestine except when

acted upon by the action of pancreatic lipase. The action of this enzyme is the most

extensively studied mechanism using natural products for their efficacy as anti-obesity

agents (Birari and Bhutani, 2007). Medicinal plants act in the gut lumen by forming a

covalent bond with the active serine site of both gastric and pancreatic lipases. The

covalent bond formed inhibits these lipases from hydrolyzing the ingested fat into

absorbable free fatty acids and monoglycerides. This decreased absorption of ingested

fat interplays into decreased caloric absorption and ultimately bring about weight loss

(Tsujita et al. 2006). Examples of plants that possess pancreatic lipase inhibitory

potentials are Ligularia fisheri (Cha, 2012), Clerodendrum phlomidis (Chidrawar, 2012)

and Nelumbo nucifera (Ahn et al. 2013). Different types of tea (e.g. green, oolong, and

black tea) have been shown to be natural sources of pancreatic lipase inhibitors (Koo

and Noh 2007; Gondoin et al. 2010).

(b) Down regulation of adipogenesis : Adipogenesis is a complex process regulated by the

expression of several hundred genes. The expression of PPAR-γ gene during

differentiation is a significant event in adipogenesis process in fat (Gao et al. 2013;

Mostafa and Labban, 2013). A variety of phytochemicals such as quercetin, kaempferol,

catechin and dietary flavonoids inherent in vegetables, fruits, green tea, herbs and

medicinal plants have been reported to inhibit the early stage of adipocyte differentiation

and the down-regulation of adipogenic transcription factors such as PPAR-γ, C/EBP-α

and SREBP-1 (Patra et al. 2015). Epigallocatechin gallate (EGCG) and tea catechins

have been reported to decrease the adipose tissue weight (Lee et al. 2009, Lee et al.

2015). Furthermore, naturally-occurring compounds have displayed apoptotic effects on

maturing 3T3-L1 preadipocytes through suppression of ERK1/2 phosphorylation,

activation of the mitochondrial pathway, AMPK activation, or anti-oxidant activity

preadipocytes, e.g. phytochemicals, such as esculetin, resveratrol, quercetin, genistein,

capsaicin, and conjugated linoleic acids (Hsu and Yen, 2006; Yang et al. 2008).

heat. It can promote weight loss because it increases the rate at which the body basis

calories. Body weight and energy expenditure are regulated by mammalian brown

adipose tissues (BAT) through the dissipation of excess energy as heat (Tseng et al.

2010). BAT plays an essential role in the control of energy balance, thus reducing the

onset of obesity. The mitochondrial uncoupling protein (UCP) dissipating energy as heat

during oxidative phosphorylation, is a major player in thermogenesis. Some medicinal

plants trigger the expression of UCP1 gene which aid in increasing energy expenditure

through thermogenesis (Chechi et al. 2014). For instance, the ethanolic extract of

Solanum tuberosum have been reported to trigger the expression of UCP3 in both BAT

and liver, which led to an appreciable reduction in the fat deposits in high-fat fed rats

(Yoon et al. 2008). Caffeine and capsaicin are some other naturally-occurring

compounds with promising weight control potentials via enhancement of energy

expenditure (Rayalam et al. 2008).

(d) Lipid Metabolism: Obesity can also be combated by stimulating triglyceride hydrolysis

in order to diminish fat stores. The oxidation of the newly released fatty acids leads to

the development of the β3-adrenergic agonists (An et al. 2010). Flavonoids in the leaf

of Nelumbo nucifera is an example of natural compounds involved in β3-adrenergic

receptor activation. The dietary supplementation with Nelumbo nucifera cause an

appreciable suppression of body weight gain in A/J mice fed a high-fat-diet (Ohkoshi et

al. 2007).

(2) Centrally acting anti-obesity substances: Restriction of food intake aids in the

maintenance of body weight. A variety of medicinal plants have been shown to exert their

effect on receptors within the central nervous system thus bringing about satiety.

(a) Neuropeptide Signaling Modulators: Adiposity-associated hormones (e.g leptin and

insulin) and gastrointestinal peptides like ghrelin communicate the stored up energy level

via central nervous system (Morton et al. 2006). The major neuronal signaling site for the

regulation of appetite hormones is made up of two neurons namely:

(1) Anorexigenic (appetite suppressing) neuropeptides, pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) and (2) Orexigenic (appetite stimulating) neuropeptide, agouti-related peptide (AgRP) and neuropeptide Y (NPY).

The expression of neuropeptide precursor proopiomelanocortin (POMC) is stimulated by neuron acted upon by leptin and insulin while the inhibition of neurons that create neuropeptide

Y (NPY) and agoutirelated protein (Ghrelin) exerts the opposite effect through the direct activation of NPY/Agrp cells thus resulting into an indirect silencing of POMC cells. The result 31

of the communication on the neurons of the hypothalamus by leptin, insulin, and ghrelin helps to control appetite and energy expenditure thus regulating the body weight. The above findings has made Leptin, Ghrelin and Neuropeptide Y promising targets/markers for obesity. Plasma leptin concentration has been reported to be by regulated by extracts of Green tea (Di Pierro et al. 2009). Also, report has shown that the administration of crude extract of adlay seed modulated the expressions of leptin and TNF-alpha and also caused the reduction in adipose tissue mass, body weights, fat size and food intake in high-fed-diet induced obese rats (Kim et al. 2004).

(b) Monoamine Neurotransmitters: Appetite control is a multifacet event involving an interrelationship between neurological and hormonal system and this helps regulate body weight. There is a school of thought that serotonin, histamine, dopamine, and their associated receptor activities are closely connected with satiety regulation. Serotonin, a monoaminergic neurotransmitter controls a variety of sensory, motor, and behavioral processes. It acts through a family of about fourteen, 5- hydroxytryptamine receptor subtypes. These receptors are promising targets for drugs that treat obesity through energy intake reduction (Zanella and

Filho, 2009). The change in central nervous system (CNS) levels of various hypothalamic neuropeptides and appetite monoamine neurotransmitters could be explored as new target sites for drugs that suppress appetite (Wynne et al. 2005). Hydroxycitric acid (HCA) derived from

Garcinia cambodia serve as a natural appetite suppressant and this is achieved by stimulation of liver gluconeogenesis. It increases the availability of 5-hydroxytryptamine or serotonin which have been implicated in the regulation of eating behavior and appetite control (Vermaak et al. 2011).

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CHAPTER THREE

Nutritional evaluation of Kedrostis africana (L.) Cogn: An edible wild plant of South Africa

This chapter has been published in Asian Pacific Journal of Tropical Biomedicine 2017;

7(5): 443–449

CHAPTER THREE

NUTRITIONAL EVALUATION OF KEDROSTIS AFRICANA (L.) COGN: AN EDIBLE WILD PLANT OF SOUTH AFRICA

Contents Pages

Abstract ...... 52

Introduction ...... 52

Materials and methods ...... 53

Result ...... 54

Discussion ...... 54

References ...... 55

Asian Pac J Trop Biomed 2017; 7(5): 443–449

HOSTED BY Contents lists available at ScienceDirect Asian Paciﬁc Journal of Tropical Biomedicine

journal homepage: www.elsevier.com/locate/apjtb

Original article http://dx.doi.org/10.1016/j.apjtb.2017.01.016 Nutritional evaluation of Kedrostis africana (L.) Cogn: An edible wild plant of South Africa

Jeremiah Oshiomame Unuoﬁn, Gloria Aderonke Otunola*, Anthony Jide Afolayan Medicinal Plants and Economic Development (MPED) Research Centre, Department of Botany, University of Fort Hare, Alice 5700, South Africa

ARTICLE INFO ABSTRACT

Article history: Objective: To evaluate the nutritional composition and elemental constituents of Received 21 Sep 2016 Kedrostis africana and their safety aspect. Received in revised form 25 Oct, 2nd Methods: Proximate parameters (moisture, ash, crude fibre, crude fat, proteins, and revised form 4 Nov 2016 carbohydrate and energy) were evaluated using ALASA methods, and elemental analysis Accepted 22 Dec 2016 by ICP-OES technique. Available online 6 Jan 2017 Results: The results from nutritional analysis showed that the tuber used for this study had a low content of crude fat and high content of ash, crude protein, crude fibre, carbohydrate and energy having the recommended dietary allowances. The tuber was rich in Keywords: major minerals Na, K, Ca and Mg, there was sufficient amount of trace elements Fe, Cu, Kedrostis africana and Zn while the anti-nutrients oxalate, phytate, alkaloids, and saponins were detected in Proximate analysis amounts that are not harmful according to Food and Agriculture Organization/World Nutritional value Health Organization. ICP-OES Conclusions: The outcome of this study suggests that this wild plant has very good Edible wild plant nutritional potentials to meet the recommended dietary allowance and it could be a cheap source of essential nutrients that may ameliorate most nutritional challenges and can contribute remarkably to the amount of nutrient intake in human and animal diet.

1. Introduction billion people especially in developing countries depend on edible wild plants in their diets [5]. Traditionally, some of With ever-increasing population pressure and fast depletion these plants are not only edible but also have high medicinal of natural resources, it has become extremely important to properties [6]. An ethnobotanical study carried out [7] revealed diversify the present day agriculture produce in order to meet that wild plants play a crucial role in sustenance of life most various human needs [1]. This diversification of agricultural especially to the rural dwellers as they depend majorly on products and their consumption habits has now shed light on a wild plants for food and medicine [8,9]. This has lead broader range of plant species, particularly those which are researchers to re-examine each and every plant with a fresh currently identified as underutilized and these could signifi- new approach towards their possible use for food or medicine. cantly contribute to improved health, nutrition, livelihoods, and Plants are generally rich in primary metabolites including pro- ecological sustainability [2]. teins, carbohydrates, vitamins, sterol and lipids, which are These edible wild plants as noted by Afolayan and Jimoh [3] essential for its survival. These primary metabolites provide the and Ali-Shtayeh et al. [4] are important sources of dietary world with food and are the basis of nutrition for the entire world nutrients in food and contribute to the proper growth and [9]. functioning of the body. Based on FAO reports, about 1 Trace elements that have been implicated in combating a variety of human ailments and disease are found mainly in *Corresponding author: Gloria Aderonke Otunola, Medicinal Plants and indigenous medicinal plants [10,11]. The functional activities of Economic Development (MPED) Research Centre, Department of Botany, University specific organs could be affected by the continuous dietary of Fort Hare, Alice 5700, South Africa. ingestion of certain elements; which can lead to their Tel: +27 74 527 6130 E-mail: [email protected] bioaccumulation beyond normal or safe levels [12]. Peer review under responsibility of Hainan Medical University. The journal Kedrostis africana (Linnaeus) Cogn. (K. africana)isa implements double-blind peer review practiced by specially invited international monoecious caudiciform plant, commonly known as “Baboon's editorial board members.

2221-1691/Copyright © 2017 Hainan Medical University. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/). 52 Jeremiah Oshiomame Unuoﬁn et al./Asian Pac J Trop Biomed 2017; 7(5): 443–449

Cucumber” with lots of herbaceous climbing or creeping vines 2.3. Determination of crude fat growing rapidly from the swollen base, resembling an English ivy with a tuber. The shoots emerge from a massive underground The powdered sample (5 g) was extracted in 100 mL of tuberous rootstock (or caudex). This tuber is a water-storage diethyl ether and then placed on an orbital shaker for 24 h. The organ that is very resistant to drought [13]. The specie is native extract was then filtered and the ether extract was collected in a to Namibia and South Africa (Eastern Cape, Free State, previously weighed (W1) clean beaker. It was thereafter equil- Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Northern ibrated with 100 mL diethyl ether and shaken for another 24 h; Cape, North West and Western Cape). K. africana tuber is the filtrate was collected in the same beaker (W1). The ether was widely used in traditional medicine as an emetic, purgative, concentrated to dryness in a steam bath and dried in an oven at diuretic, anti-dropsy and to treat syphilis [14]. Also, a decoction 40–60 C and the beaker was reweighed (W2). The crude fat from the crushed fresh bulb is taken twice daily for the content was calculated as: management of obesity [15,16]. Keeping in mind its medici- W2 − W1 nal importance, the present investigation was undertaken to % Crude fat = × 100 ascertain the nutritive potential of the specie harvested from the Weight of original sample Eastern Cape of South Africa, which has been lacking in the literature. 2.4. Determination of crude fibre 2. Materials and methods A modification of the method described by Aina et al. [18] was used where 2 g of sample was digested by boiling with The tubers of K. africana used for this study were harvested 100 mL of 1.25% sulphuric acid solution for 30 min, then in August 2015 at Fort Beaufort in the Amathole District Mu- filtered under pressure. The residue was rinsed four times with nicipality, Eastern Cape, South Africa. This area lies at Latitude boiling water. This process was repeated on the residue using 3243028.6600 and Longitude 263405.8800. The plant's identity 100 mL of 1.25% NaOH solution. The final residue was then was validated by Mr. Tony Dold of Selmar Schonland Herbar- dried at 100 C, cooled in a desiccator and weighed (C1). It ium, Rhodes University, South Africa, and a voucher specimen was thereafter incinerated in a muffle furnace at 550 C for (Unuofin Med, 2015/2) was prepared and deposited in the Giffen 5 h, then transferred to cool in a desiccator and reweighed Herbarium, University of Fort Hare. The bulb was rinsed with (C2). The percentage crude fibre was calculated as: deionised water and gently blotted with paper towel, chopped into small bits, oven-dried (LABOTEC, South Africa) at 55 C for 72 h until constant weight was achieved and then ground into C2 − C1 ® % Crude fibre = × 100 powder (Polymix PX-MFC 90D Switzerland). Weight of original sample

2.1. Determination of moisture content 2.5. Determination of crude protein The moisture content was determined as published methods [17]. An empty weighing vessel was oven dried at 105 C for one The powdered sample (2 g) was digested in a Kjeldahl flask hour, cooled in a desiccator and weighed (W1). A dry sample by boiling with 20 mL of concentrated H2SO4 and a digestion weighing (2.000 ± 0.001) g (W2) was put into the vessel and tablet (catalyst) until the mixture was clear. The digest was oven dried at 105 C until constant weight was attained. This filtered and made up to mark in a 250 mL volumetric flask, was then cooled in a dessicator, after which it was weighed then distilled. The aliquot plus 50 mL of 45% sodium hy- (W3). The percentage moisture was calculated as: droxide solution was transferred into a 500 mL round bottom flask and distilled. 150 mL of the distillate was collected into a W2 − W3 % Moisture content = × 100 flask containing 100 mL 0.1 N HCl. This was then titrated W2 − W1 against 2.0 mol/L NaOH using methyl orange as indicator. The end point was indicated by a colour change to yellow. 2.2. Measurement of ash content The % nitrogen content was calculated as: ½ðmL standard acid × N of acidÞ – ðml blank × N of baseÞ The ash content was determined as described methods [17].A – ð Þ : porcelain crucible marked with a heat resistant marker was dried ml std base × N of base ×14007 at 105 C for 1 h, left to cool in a desiccator and weighed (W1). Weight of sample in grams Then 2 g of the ground sample was placed in the previously weighed crucible and reweighed (W2). The crucible with its where, N = normality, percentage crude protein was obtained by content was then ashed first at 250 C for an hour and at multiplying the nitrogen value by a factor of 6.25. % crude 550 C for 5 h in a muffle furnace. The samples were allowed protein = Nitrogen in sample × 6.25. to cool in a desiccator and then weighed (W3). The percentage ash was calculated as: 2.6. Determination of carbohydrate

The carbohydrate content was calculated by subtracting the W2 − W3 total crude protein, crude ﬁbre, ash and lipid from the total dry % Ash content = × 100 W2 − W1 matter as:

53 Jeremiah Oshiomame Unuoﬁn et al./Asian Pac J Trop Biomed 2017; 7(5): 443–449

% Total carbohydrate = 100 − ð% Moisture content + % Total Ash remaining solution was collected and heated to evaporation in a + % crude Fat + % Crude Fibre water bath, then dried to constant weight at 40 C in an oven. + % Crude ProteinÞ The saponin content was calculated using the equation: Weight of residue % Saponin content = × 100 2.7. Determination of energy content Weight of original sample

The estimated energy value in kilocalorie (Kcal/100 g) was 2.11. Determination of alkaloids calculated by summing the multiplied values for crude protein, crude lipid and carbohydrate respectively, using the factors The described method [22] was adopted. Briefly, 5 g of plant (4 kcal, 9 kcal and 4 kcal) as: extract was mixed with 200 mL of 10% acetic acid in ethanol. Energy valueðkcal=100 gÞ = ½ðcrude protein × 4Þ + ðcrude fat × 9Þ The mixture was covered and allowed to stand for 4 h. This fi fi + ðtotal carbohydrate × 4Þ was ltered and the ltrate was concentrated on a water bath to a quarter of its original volume. Concentrated ammonium hydroxide was added in drops to the extract until precipitation 2.8. Determination of oxalate content (cloudy fume) was completed. The solution was allowed to settle, washed with dilute ammonium hydroxide and then filtered. The residue collected was dried and weighed and the The modified titration method [18,19] was used to determine alkaloid content was calculated using the equation: the oxalate content of the plant. The pulverized sample (1 g) fl was weighed into a conical ask; 75 mL of 3 mol/L H2SO4 Weight of precipitate % Alkaloid = × 100 was added and stirred with a magnetic stirrer for an hour. This Weight of original sample was filtered and 25 mL of the filtrate was collected and heated to 80–90 C. This filtrate was kept above 70 C at all times. The hot aliquot was titrated continuously with 0.05 mol/L of 2.12. Elemental analysis KMnO4 until the end point revealed by a light pink colour which persisted for 15 s was reached. The method described [23] using Inductively Coupled The oxalate content was calculated by taking 1 mL of Plasma-Optical Emission Spectrometer (ICP-OES; Varian 0.05 mol/L of KMnO4 as equivalent to 2.2 mg oxalate. 710–ES series, SMM Instruments, Cape Town, South Africa) was used to determine the elemental constituent of the sample. 2.9. Determination of phytate content All analyses were carried out in triplicates.

Phytic acid was determined as described [20]. The sample 2.13. Statistical analysis of data (2 g) was weighed into a flask, 100 mL of 2% HCl was added and allowed to stand for 3 h, after which it was filtered. All experiments were carried out in triplicates and the data 25 mL of the filtrate was placed in a separate 250 mL conical expressed as mean ± SD using the Microsoft Excel 2010 flask with 5 mL of 0.3% ammonium thiocyanate solution as spreadsheet. indicator. 53.5 mL of distilled water was added to give the desired acidity. This was then titrated with standard iron III 3. Results chloride solution (0.001 95 g of iron per mL) until a brownish yellow colour persisted for 5 min. Phytic acid was calculated as: 3.1. Proximate composition Phytic acid (%) = titre value × 0.001 95 × 1.19 × 100 The result of the proximal content of K. africana tuber is presented in Table 1. The moisture content was low (3.77 ± 0.14)%, with high ash value (8.94 ± 0.28)%. The per- 2.10. Determination of saponins centage fibre content was relatively high (25.52 ± 0.23)%, crude fat content was very low (1.12 ± 0.42)%, while crude protein Saponin content was estimated as described [21]. Briefly, 5 g was (6.95 ± 0.11)%. The carbohydrate content was high of the pulverized plant sample was added to 50 mL of 20% (46.36 ± 0.23)% and the overall estimated energy value of the ethanol, kept on a shaker for 30 min and then heated in a whole plant of K. africana was (223.37 ± 0.88) Kcal/100 g. water bath at 55 C for 4 h. The resulting mixture was filtered and the residue re-extracted with another 200 mL of 20% Table 1 aqueous ethanol. The filtrates were combined and reduced to Proximate composition of K. africana. 40 mL in a water bath at 90 C. The concentrate was transferred Parameters % into a separating funnel, 20 mL of diethyl ether was added, and shaken vigorously. The ether layer which was the upper layer Moisture 3.77 ± 0.14 was discarded and the aqueous (bottom) layer retained in a Total ash 16.28 ± 0.06 Crude fat 1.12 ± 0.42 beaker. The retained layer was re-introduced into a separating Crude fibre 25.52 ± 0.23 funnel and 60 mL of n-butanol was added and shaken vigor- Crude protein 6.95 ± 0.11 ously. The butanol extract which is the upper layer was retained Carbohydrate 46.36 ± 0.23 while the bottom layer was discarded. The butanol layer was Energy value (Kcal/100 g) 223.37 ± 0.88 washed twice with 10 mL of 5% aqueous sodium chloride. The Values are expressed as mean ± SD, n =3.

54 Jeremiah Oshiomame Unuoﬁn et al./Asian Pac J Trop Biomed 2017; 7(5): 443–449

3.2. Anti-nutrient factors flavours [29]. Excess intake of dietary fat is a major cause of cardiovascular diseases, cancer, and aging. It has been The antinutritional composition of K. africana is shown in suggested that 1%–2% of caloric energy from fat is best for Table 2. The saponin content was (1.94 ± 0.42)%, alkaloid was healthy living [30]. In this regard, the low crude fat content of (0.30 ± 0.08)%, phytate was (2.42 ± 0.17)%, while oxalate K. africana implies that it could prevent certain chronic content was (0.298 ± 0.150)%. ailments in humans associated with lipids. This perhaps justifies its folkloric use in the management of obesity [15]. Dietary proteins are pivotal in the manufacturing and safe- Table 2 guarding of certain organic materials necessary for the smooth Anti-nutrient composition of K. africana. functioning of the human body [31]. The relatively high protein Anti-nutrient % content of K. africana could make it a useful supplement to Phytic acid 2.42 ± 0.17 diets with little amount of proteins most especially grains. The Oxalate 0.28 ± 0.15 high carbohydrate content makes it a rich source of energy Saponins 1.94 ± 0.42 and this could be used to enrich the energy content of diets Alkaloids 0.30 ± 0.08 [29]. The low overall estimated energy value of the whole Each value represents the mean ± SD of three determinations on dry plant of K. africana can be attributed to its low crude fat and weight basis. moisture levels. This corroborates the fact that K. africana as a low energy food source may be very helpful in weight 3.3. Elemental content management programmes as used by traditional healers. The saponin content of K. africana was low and within the safe The result for the mineral analysis of K. africana whole plant limit, since an amount below 10% is not hazardous to the body is presented in Table 3. Calcium content (2505.00 mg/100 g) [32]. High saponin levels in human and animal diets have been was highest compared to other minerals analysed. The potassium implicated in growth impairment, reduction in bioavailability of content was 2225.00 mg/100 g, phosphorus was 240 mg/100 g, nutrients and inhibition of biochemical reactions that facilitate magnesium content was 485 mg/100 g, sodium 430.00 mg/ breakdown of ingested proteins [33]. 100 g; while iron content was 89.9 mg/100 g, zinc 4.80 mg/ Alkaloids are one of the most efficient therapeutic bioactive 100 g, manganese 3.1 mg/100 g and copper content was 0.1 mg/ substances in plants. For instance, consumption of high tropane 100 g. alkaloids will cause rapid heartbeat, paralysis and in fatal case, lead to death. Uptake of a high dose of tryptamine alkaloids will [34] Table 3 lead to the staggering gait and death . Other toxic action Mineral composition of K. africana. includes disruption of the cell membrane in the gastrointestinal tract [35]. The alkaloid content recorded in this study was quite Mineral mg/100 g DW low, mitigating the fear of anti-nutrient activity. Calcium 2505.00 ± 21.21 The phytate content of K. africana was low. A dietary phy- Magnesium 485.00 ± 7.07 tate content of 1%–6% over a long period decreases the Potassium 2225.00 ± 35.36 bioavailability of mineral elements in monogastric animals [36]. Phosphorous 240.00 ± 0.00 Sodium 430.00 ± 14.14 The excessive intake of phytate rich diets is associated with Zinc 4.80 ± 0.28 nutritional diseases such as rickets and osteomalacia in Copper 0.10 ± 0.00 children and adults respectively. However, this anti-nutrient Manganese 3.10 ± 0.00 could easily be removed by soaking, boiling or frying [37]. Iron 89.90 ± 0.85 The presence of oxalate in foods causes irritation in the Each value represents the mean ± SD of three determinations on dry mouth and could decrease the absorption of calcium and in- weight basis. crease the formation of kidney stone [38,39]. The concentrations of anti-nutrients (saponin, oxalate, phytate and alkaloids) 4. Discussion recorded in this study were however within the safe limit and may not elicit toxic effect when consumed especially when The stability and shelf-life of any food component is deter- thermally treated before use. mined by its moisture content [24]. The low moisture content Minerals are considered to be essential in human nutrition for implies that K. africana may have a long shelf life and the overall mental and physical well being, as well as important reduced microbial contamination [24]. The high ash value constituents of bones, teeth, tissues, muscles, blood, and nerve suggests that the plant is a rich source of mineral since ash cells. content is an indication of mineral composition. Dietary fiber The recommended daily allowance (RDA) of calcium for encourages the proliferation of advantageous organisms in the adults is 1000 mg [40]. Therefore K. africana is capable of intestinal flora and lessens the risk of colon cancer [25]. The contributing almost three times the RDA for calcium. Calcium relatively high fibre content is an indication that the intake of is needed for growth and maintenance of bones, teeth, and K. africana could aid digestion, facilitate peristaltic movement muscle, and as such may be used as supplements in diets low in and thus prevent constipation [26]. High fibre intake could lead calcium ion. Calcium plays a vital role in muscle contraction; to a decline in the incidence of certain diseases associated strong bones, neurological function and also to surmount the with metabolic disorders [27]. However when fibre is present problems of high blood pressure, heart attack, premenstrual in abundance, it could negatively affect the absorption of syndrome, colon cancer and osteoporosis in old age [41,42]. certain minerals which are beneficial to the body e.g. iron [28]. The RDA for potassium in adults is 4700 mg [43]. Dietary lipids improve the taste of food by preserving its K. africana is able to contribute 47% (almost half) of this

55 Jeremiah Oshiomame Unuofin et al./Asian Pac J Trop Biomed 2017; 7(5): 443–449 amount if included in the diet. Potassium is effective in reducing The copper content of K. africana is however below the hypertension, maintaining the cardiac rhythm and is also crucial RDA of 0.7, or 1.1 mg/day required for children and adults, in many physiological functions [44]. It regulates heartbeat, respectively [52]. Copper is involved in erythropoiesis, eryth- neurotransmission and water balance of the body [45]. rocyte function and regulation of red blood cell survival. The magnesium content of this plant is high, and could However, high concentration of copper can lead to diarrhoea, contribute about 1.2 times more than the RDA of 450 mg/day epigastric pain and discomfort, blood in the urine, liver required for humans [46]. Magnesium enhances the beta cells damage, hypotension and vomiting [62]. functions thus preventing onset of diabetes and hypertension The study revealed that K. africana had high fibre, calcium, [47]. Also, it has been implicated as necessary for a number of iron, manganese, and magnesium contents. The anti-nutrients enzymatic reactions such as oxidative metabolism of nutrients, content were within acceptable limits and hence may not inter- cell constituents synthesis, transmission of nerve impulses, fere with absorption of other nutrients. The level of these anti- body temperature regulation, detoxification, energy production nutrients can also be reduced or totally eliminated by prepara- and the formation of strong bones and teeth [48]. tion techniques such as soaking, blanching, steaming, boiling K. africana is capable of contributing up to 29% of the RDA and cooking. K. africana is rich in many macro and micro nu- of sodium for adults in relation to the 1500 mg recommended trients and can therefore serve as a supplement to prevent many for adults [49]. A ratio of sodium to potassium ion less than one mineral deficiencies. K. africana should therefore be considered (Na+/K+ < 1) has been reported to be suitable for reducing high a plant with great potential in the food, nutritional and phar- blood pressure. This therefore suggests that the plant could be a maceutical industries. Further studies on toxicity of K. africana good dietary supplement for hypertensive patients. Sodium is are on-going to ascertain its possible adverse effects and to involved in the maintenance of osmotic pressure of the body confirm some of the ethno-pharmacological claims. fluids, irritability of muscles and cell permeability. It plays an important role in the maintenance of membrane potentials, Conflict of interest statement transmission of nerve impulses and also in the absorption of monosaccharides, amino acids, pyrimidines, and bile salts [50]. We declare that we have no conflict of interest. Phosphorus is an important mineral that aids the absorption of calcium which is required for growth, maintenance of bones, Acknowledgments teeth and muscles [51]. The potassium content of K. africana is within the RDA of 200–1000 mg/day for children and adults, The authors wish to acknowledge the financial support of the respectively [52]. Phosphorus in association with calcium National Research Foundation (NRF 85294), South Africa. contributes to bones and teeth reinforcement especially in children and nursing mothers [53]. References The high amount of iron found in K. africana indicates that the plant could be a good source of dietary iron and could [1] Deb CR, Jamir NS, Sungkumlong O. A study on the survey and contribute almost 5 times more than the RDA of 18 mg/day documentation of underutilized crops of three districts of Nagaland, needed by adults [54] to overcome the nutritional deficiency of India. J Glob Biosci 2013; 2(3): 67-70. iron if used as a supplement in the diet. Iron deficiency is a [2] Romojaro MA, Botella C, Pretel MT. Nutritional and antioxidant common nutritional problem affecting many people world- properties of wild edible plants and their use as potential ingredients in the modern diet. Int J Food Sci Nutr 2013; 64(8): 944-52. wide; primarily, it occurs as a result of chronic bleeding, [3] Afolayan AJ, Jimoh FO. Nutritional quality of some wild leafy fi infections, inadequate intake of bioavailable iron, de ciencies vegetables in South Africa. 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Ethnobot Leafl 2010; 14: 751-8. healing, reduce prostate enlargement and stress [58]. [8] Chen B, Qiu Z. Consumers' attitudes towards wild edible plants. A The manganese content can contribute about 1.35 times the case study of Noto Peninsula, Ishikawa, Prefecture, Japan. Int J Res recommended RDA value [59]. Manganese plays a crucial role in 2011; 5: 1-16. skeletal growth and development, and also functions with [9] Karuna SV, Vijaya SK. Nutritive assessment of different plant parts of Carica papaya Linn of Jabalpur region. J Nat Prod Plant vitamin K in the formation of prothrombin. It acts as a Resour 2014; 4(1): 52-6. catalyst and co-factor in many enzymatic processes, involved [10] Shirin K, Imad S, Shafiq S, Fatima K. 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Proxi- Normal range of human dietary sodium intake: a perspective based mate and anti-nutritional constituents of Abelmoschus esculentus on 24-hour urinary sodium excretion worldwide. Am J Hypertens grown in Fadaman Kubanni, Zaria, Kaduna State. Niger J Sci Res 2013; 26(10): 1218-23. Rep 2014; 3: 2015-27. [50] Soetan CO, Olaiya CO, Oyewole OE. The importance of mineral [29] Anita BS, Akpan EJ, Okon PA, Umoren IU. Nutritive and anti- elements for humans, domestic animals and plants: a Review. Afr J nutritive evaluation of sweet potatoes (Ipomoea batatas) leaves. Food Sci 2010; 4(5): 200-22. Pak J Nutr 2006; 5: 166-8. [51] Veum TL. Phosphorus and calcium nutrition and metabolism. In: [30] Aruah BC, Uguru MI, Oyiga BC. Nutritional evaluation of some Vitti DMSS, Kebreab E, editors. Phosphorus and calcium utili- Nigerian pumpkins (Cucurbita spp.). Fruit Veg Cereal Sci Bio- zation and requirements in farm animals. UK: Oxfordshire; 2010, technol 2011; 5: 64-71. p. 94-111. 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CHAPTER FOUR

Essential oil composition, nutrient and antinutrient analysis of Vernonia mespilifolia Less.

This chapter has been published in Research Journal of Botany

CHAPTER FOUR Essential oil composition, nutrient and antinutrient analysis of Vernonia mespilifolia Less.

Contents Pages

Introduction ...... 61

Materials and methods ...... 62

Results and discussion ...... 63

Conclusion ...... 66

References ...... 66

OPEN ACCESS Research Journal of Botany

ISSN 1816-4919 DOI: 10.3923/rjb.2017.38.45

Research Article Essential Oil Composition, Nutrient and Anti-nutrient Analysis of Vernonia mespilifolia Less.

Jeremiah Oshiomame Unuofin, Gloria Aderonke Otunola and Anthony Jide Afolayan

Medicinal Plants and Economic Development Research Centre, Department of Botany, University of Fort Hare, 5700 Alice, South Africa

Abstract Objective: This study aimed to evaluate the essential oil, nutrients and anti-nutrient content of Vernonia mespilifolia an indigenous medicinal plant used traditionally by the people of Eastern Cape, South Africa for the management of obesity. Methodology: Proximate parameters (moisture, ash, crude fats, proteins, crude fibers, carbohydrates and energy values) and mineral analysis (K, Na, Ca, Fe, P and Mg etc.) were evaluated using standard techniques. Essential oil was extracted using a rapid solvent-free microwave extraction method and analyzed using Gas Chromatography-Mass Spectrometer (GC-MS). Results: The results revealed that carbohydrate content was (46.66±0.44%), protein (10.75±0.08%), moisture (3.45±0.29%), fat (0.97±0.25%) and fibre (29.24±0.67%), while the gross total energy was 238.37±0.87 kcal/100 g. The minerals detected in appreciable quantity ranged from magnesium (1.55 mg/100 g) to potassium (2175.00 mg/100 g). The anti-nutrients (phytate, oxalate, saponins and alkaloids) content was relatively low compared with those of most edible plants and are not likely to cause any significant interference with nutrient absorption. The GC-MS analysis of the essential oils showed higher proportion of 2,5-dimethylhexa-2,4-diene, "-gurjunene, kaur-16-ene, acetamide, N-(3-nitrophenyl)-2,2-dichloro, "-elemene. Conclusion: The nutrients, mineral and essential oil contents of V. mespilifolia could be a good addition to the diet besides its medicinal values.

Key words: Proximate parameters, essential oil, anti-nutrients, GC-MS, mineral, Vernonia mespilifolia.

Received: November 04, 2016 Accepted: February 09, 2017 Published: March 15, 2017

Citation: Jeremiah Oshiomame Unuofin, Gloria Aderonke Otunola and Anthony Jide Afolayan, 2017. Essential oil composition, nutrient and anti-nutrient analysis of Vernonia mespilifolia Less. Res. J. Bot., 12: 38-45.

Corresponding Author: Gloria Aderonke Otunola, Medicinal Plants and Economic Development (MPED) Research Centre, Department of Botany, University of Fort Hare, 5700 Alice, South Africa Tel: +27 (0) 40602 2320

Copyright: © 2017 Jeremiah Oshiomame Unuofin et al. This is an open access article distributed under the terms of the creative commons attribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original author and source are credited.

Competing Interest: The authors have declared that no competing interest exists.

Data Availability: All relevant data are within the paper and its supporting information files.

61 Res. J. Bot., 12 (2): 38-45, 2017

INTRODUCTION Proximate analysis: The proximate parameters (moisture, dry matter, ash, crude fats, proteins and fibers, nitrogen, Vernonia mespilifolia Less. popularly known as carbohydrates and energy values) were determined using Uhlunguhlungu (Xhosa) among the indigenous people of the Association of Official Analytical Chemists Methods10,11. Nkonkobe Municipality of the Eastern Cape of South Africa is Determination of moisture content was done by drying one of the five Southern African species of the Vernonia family samples in oven (LABOTEC, South Africa) at 110EC until that is endemic or near-endemic to this subcontinent1. It is a constant weight was attained12. Nitrogen estimation was climbing shrub that is 0.6-9.0 m tall, with pinnately-veined carried out using the micro-Kjeldahl (BUCHI, KjelFlex K-360, leaves, epaleate receptacle with obtuse involucral bracts and Switzerland) method with slight modification10. The crude white to violet florets1. Vernonia mespilifolia is wide-spread proteins were subsequently calculated by multiplying the in the Eastern Cape, Kwazulu-Natal, Limpopo, Mpumalanga nitrogen content by a factor of 6.2510. The energy value and Western Cape provinces of South Africa2. It is used for estimation was done by summing the multiplied values for ethno-medicinal management of weight loss and crude protein, crude fat and carbohydrate at water factors hypertension3 and for the treatment of heart water disease in (4, 9 and 4) respectively. Crude fats were determined by goats4. In some parts of Africa, the popular sister species, such Soxhlet apparatus using n-hexane as a solvent. The ash values as V. amygdalina, V. calvaona and V. colorata have been were obtained by heating samples at 550EC in a muffle thoroughly investigated with respect to human nutrition and furnace (E-Range, E300-P4, MET-U-ED South Africa) for 3 h. The medicinal potentials for their hypoglycemic and carbohydrate content was determined by subtracting the total hypolipidemic effects, as well as antimalarial, anthelminthic, crude protein, crude fiber, ash content and crude fat from the anti-diabetic and antitumorigenic activities5-8. Despite the total dry matter12. Crude fiber was estimated by acid-base popular use of V. mespilifolia in folk and traditional medicine, digestion with 1.25% H2SO4 (v/v) and 1.25% NaOH (w/v) 11 the plant is not much studied scientifically for its nutritive and solutions . biological properties. Essential oils are aromatic and largely volatile Anti-nutritive components compounds which are commonly extracted by using solvent Determination of oxalate content: The modified method of free extraction and hydrodistillation method. The solvent free Agbaire13 was used to determine the oxalate content of the microwave-assisted essential oil extraction method was used plant. Approximately 1 g of the pulverized sample was because of its efficiency to prevent disintegration of fragile weighed into a conical flask, 75 mL of 3 M H2SO4 was added volatile components of essential oils9. Despite its renowned and stirred with a magnetic stirrer for an hour. This was filtered medicinal potential, little or no report is known about its and 25 mL aliquot of the filtrate was collected and heated to nutritional benefits hence this study aimed to analyze the 80-90EC. This filtrate was kept above 70EC at all times. The hot proximate parameters, anti-nutritional content, mineral aliquot was titrated against 0.05 M of KMnO4 until an compositions and essential oil constituent of V. mespilifolia extremely faint pale pink colour persisted for 15-30 sec. The used in the management of obesity in Eastern Cape, South oxalate content was calculated by taking 1 mL of 0.05 M of

Africa. KMnO4 as equivalent to 2.2 mg oxalate.

MATERIALS AND METHODS Determination of phytic acid: Phytic acid was determined as described by Damilola et al.14. Approximately 2 g of the The whole plant parts (leaves, flowers, stems and roots) sample was weighed into a 250 mL conical flask. of V. mespilifolia used for this study were collected in August Approximately 100 mL of 2% HCl was used to soak the sample 2015 from the wild at Dimbaza village near Alice in the Eastern for 3 h and then filtered through Whatman No. 1 filter paper. Cape Province of South Africa. The plant was authenticated by Approximately 25 mL aliquot of the filtrate was placed in a Mr. Tony Dold of Selmar Schonland Herbarium, Rhodes separate 250 mL conical flask and 5 mL of 0.3% ammonium University, South Africa and a voucher specimen (Unuofin thiocyanate solution was added indicator. Approximately Med, 2015/1) was prepared and deposited in the Giffen 53.5 mL of distilled water was added and this was then titrated Herbarium, University of Fort Hare. The whole plant was rinsed with standard iron III chloride solution which contains with deionized water and gently blotted with paper towel to 0.00195 g of iron per milliliter until a brownish yellow colour remove the water, oven-dried (LABOTEC, South Africa) at persisted for 5 min. Phytic acid was calculated as Eq. 1: 40EC for 72 days until constant weight was achieved, then ground into powder (Polymix® PX-MFC 90D Switzerland). Phytic acid (%) = Titre value×0.00195×1.19×100 (1)

62 Res. J. Bot., 12 (2): 38-45, 2017

Determination of saponin: Saponin content was determined with Perkin Auto-Sampler with the following parameters: as described by Obadoni and Ochuko15. Briefly, 5 g of the plasma flow rate (15 L minG1), nebulizer flow rate (0.8 L minG1), pulverized plant sample was added to 50 mL of 20% ethanol, RF power (1500 W), auxiliary flow rate (0.2 L minG1), sample kept on a shaker for 30 min and then heated in a water bath flow rate (1.25-2.50 L minG1), torch position (-3) for aqueous at 55EC for 4 h. The resulting mixture was filtered and the samples and 15 sec equilibration. residue re-extracted with another 200 mL of 20% aqueous ethanol. The filtrates were combined and reduced to 40 mL in Solvent Free Microwave Extraction (SFME) of essential oils: a water bath at 90EC. The concentrate was transferred into a The SFME was carried out with a microwave essential oil separating funnel, 20 mL of diethyl ether was added and system (MILESTONE Microwave Laboratory Systems, Apollo shaken vigorously. The ether layer which was the upper layer Scientific, South Africa) with a maximum delivery power of was discarded and the aqueous (bottom) layer retained in a 900 W variables in 10 W increments and 650 nm wavelength. beaker. The retained layer was re-introduced into a separating During experiment, time, pressure and power were funnel and 60 mL of n-butanol was added and shaken controlled with the “Easy-WAVE” software. Fresh samples of vigorously. The butanol extract which is the upper layer was V. mespilifolia (100 g each) were heated using a fixed power retained while the bottom layer was discarded. The butanol of 400 W for 30 min at 100EC. The essential oils were collected, layer was washed twice with 10 mL of 5% aqueous sodium dried over anhydrous sodium sulphate and stored at 4EC until chloride. The remaining solution was collected and heated to needed. Extractions were performed at least three times and evaporation in a water bath, then dried to constant weight at the mean values are presented. 40EC in an oven. The saponin content was calculated using the Eq. 2: Gas chromatography mass spectrometry (GC-MS): The GC-MS analysis was performed using an Agilent 7890B GC Weight of residue system equipped with an Agilent 5977A mass selective Saponin content (%) = ×100 (2) Weight of original sample detector (Chemetrix, Pty, Ltd, Agilent Technologies, DE, Germany) and a Zebron-5MS (cross-linked 5% phenyl Determination of alkaloids: The alkaloid content was methylpolysiloxane) column (ZB-5MS 30 m×0.25 mm determined according to the method of Omoruyig et al.16. ×0.25 μm). The following column and temperature conditions Briefly, 5 g of plant extract was mixed with 200 mL of 10% were used: GC grade helium was used as carrier gas at a flow acetic acid in ethanol. The mixture was covered and allowed rate of 2 mL minG1 and splitless 1 mL injections was used. The to stand for 4 h. This was filtered and the filtrate was injector and source temperatures were both set at 280EC. concentrated on a water bath to one-fourth of its original Initial oven temperature was 70EC. This was then ramped at volume. Concentrated ammonium hydroxide was added in 15EC minG1 to 120EC, then ramped at 10EC minG1 to 180EC drops to the extract until precipitation (cloudy fume) was and then ramped at 20EC minG1 to 270EC and finally held at completed. The solution was allowed to settle, washed with this temperature for 3 min. The data obtained was gathered dilute ammonium hydroxide and then filtered. The residue with Chem Station. Identification of the components of collected was dried and weighed and the alkaloid content was essential oils was done by comparison of mass spectra calculated using the Eq. 3: obtained with those stored in NIST11.L library, PubChem Project (https://pubchem.ncbi.nlm.nih.gov/) and DrugBank Weight of precipitate (www.drugbank.ca/) to identify the known pharmacological Alkaloid (%) = 100 (3) Weight of original sample properties associated with these compounds..

Macro and micro-nutrients analysis: The elemental profile of Statistical analysis: All experiments were performed in V. mespilifolia was analyzed as described by Bvenura and triplicates and the results expressed as Mean±SD using the Afolayan17. The dried homogenized sample (0.5 g) was taken Microsoft Excel 2010 spreadsheet. in a Kjeldahl tube (250 mL) and digested with 20 mL of 98% sulphuric acid (Sigma Aldrich) at 370EC to a colorless liquid. RESULTS AND DISCUSSION The resultant liquid was diluted with distilled water up to 100 mL and filtered using Whatman-42 filter paper. This was Proximate composition: The result for the proximal content then analyzed using Inductively Coupled Plasma Emission of V. mespilifolia is presented in Table 1. The moisture content Spectrometer (ICP-OES DV 7300, Perkin Elmer, USA) equipped was low (3.45±0.29%), this is a pointer that the plant will have

63 Res. J. Bot., 12 (2): 38-45, 2017

Table 1: Proximate composition of Vernonia mespilifolia Less. Table 3: Mineral composition of Vernonia mespilifolia Less. Parameters Composition (%) Mineral elements Composition (mg/100 g) Moisture content 3.45±0.29 Calcium 485.00±7.07 Total ash 8.94±0.28 Magnesium 140.00±0.00 Crude fat 0.97±0.25 Potassium 2175.00±7.07 Crude fibre 29.24±0.67 Phosphorous 400.00±0.00 Crude protein 10.75±0.08 Sodium 570.00±14.14 Carbohydrate 46.66±0.44 Zinc 4.40±0.07 Energy value (kcal/100 g) 238.37±0.87 Copper 1.55±0.07 Values are expressed as Mean±SD, n = 3 Manganese 4.70±0.14 Iron 26.5±0.49 Values are expressed as Mean±SD, n = 3 Table 2: Anti-nutrient composition of Vernonia mespilifolia Less. Parameters Values (%) Phytic acid 3.23±0.35 have been connected with gastroenteritis, manifested by Oxalate 0.29±0.05 diarrhoea, dysentery and haemolysis of red blood cells of Saponins 3.28±0.21 rats22,23. According to Ridout et al.24 and Umaru et al.25 Alkaloids 0.62±0.03 saponins protect plants from fungal and insect attacks but in Values are expressed as Mean±SD, n = 3 humans and animals saponin reduces body cholesterol by a protracted storage period and thus it would not be liable to preventing its reabsorption and suppresses rumen protozoan microbial spoilage18. The moisture content is an essential by reacting with cholesterol in the protozoan cell membrane thereby causing it to lyse. aspect to consider in handling, safeguarding and sustenance The alkaloid content was 0.62±0.11%. Alkaloids are one of foodproducts18. The high ash content (8.94±0.28%) hints of the most efficient therapeutic bioactive substances in that V. mespilifolia has a high mineral. The study revealed plants. Some alkaloids stimulate the nervous system, others that V. mespilifolia has high fibre content (29.24±0.67%) and can cause paralysis, elevate blood pressure or lower it26. as such its ingestion could aid in digestive processes and The phytate content of the plant was 3.23±0.35%. also reduce the absorption of cholesterol which has been According to Oke27, a phytate diet of 1-6% over a long period implicated in the onset of cardiovascular diseases and decreases the bioavailability of mineral elements in cancer19. Furthermore, it could provide assistance to the monogastric animals. It also forms insoluble complexes with gastrointestinal tract in the area of providing bulk to stool and a variety of minerals most especially the divalent ones such as lubrication of the colon20. The crude fat level of V. mespilifolia calcium, copper, manganese etc., thereby lessening the was 0.97±0.25%. This is quite low and could be advantageous accessibility of these nutrients28. This indicates that the if made a part of the diet for individuals suffering from consumption of large amounts of Vernonia mespilifolia may overweight or obesity. This perhaps justifies the already locally have adverse effects on human health. However, this established use of the plant in the management of obesity3. anti-nutrient could easily be removed by blanching, boiling or The crude protein content of Vernonia mespilifolia was found frying29. to be 10.75±0.08%. This value is relatively high and could The oxalate content was 0.29±0.05%. The presence of complement protein from cereals and other plant foods that oxalate in foods causes irritation in the mouth and interferes are known to be low in protein in the diet of consumers. The with absorption of divalent minerals particularly calcium by carbohydrate content of 46.66±0.44% suggests that the plant forming insoluble salts30,31. This renders calcium unavailable is a rich source of energy and could be used to enrich the for normal physiological and biochemical roles, such as the 21 energy content of diets . The overall estimated energy of the maintenance of strong bone, teeth, cofactor in enzymatic whole plant of V. mespilifolia was 238.37±0.87 kcal/100 g reaction, nerve impulse transmission and clotting factor in (Table 1). This energy level is low due to the low crude fat and the blood. In addition, high oxalate intake can result in moisture level and attests to the fact that V. mespilifolia is a hyperoxaluria thereby increasing the risk of kidney stones32,33. low energy food source and as such may be very helpful in The concentrations of anti-nutrients (saponin, oxalate, phytate weight management program as used by traditional healers. and alkaloids) recorded in this study are within tolerable limit and may not elicit toxic effect when consumed especially Anti-nutrient factor: The result of anti-nutrient analysis of when the specie is thermally treated before use. Vernonia mespilifolia is shown in Table 2. The saponin content (3.28±0.21%) was found to be within the safe limit, since Elemental content: The result for the mineral analysis of saponins at levels <10% in a diet is said to be harmless to the Vernonia mespilifolia presented in Table 3, showed that body22. However, in humans and animals high saponin levels potassium content (2175.00 mg/100 g) was higher in the plant

64 Res. J. Bot., 12 (2): 38-45, 2017 compared to other minerals analyzed. The recommended blood formation. It is crucial in energy production, daily allowance (RDA) of potassium for adults is 4700 mg34. neurotransmitter synthesis and maintaining a stable immune Therefore, V. mespilifolia is able to contribute almost half of system42. Iron down-regulates genes such as hepcidin, LXRá the RDA for potassium, this is an indication that the plant is a and FPN which are basic immunological factors thereby fairly good source of potassium. Potassium is the main reducing cellular Reactive Oxygen Species (ROS), tissue intracellular cation in the human body required for vital damage, lipid retention and inflammation. This infers the role cellular processes. It is involved in regulating acid-base of iron as a therapeutic agent against inflammation and balance, blood pressure, cell membrane function and basic atherosclerotic conditions43. The manganese content cellular enzymatic reaction35. The sodium content 4.7 mg/100 g is high and can contribute up to 94% RDA (570.00 mg/100 g) of the plant is high, contributing 95% RDA proportion for children and adults in relation to the 1 44 proportion for adults in relation to the 600 mg RDA for an 2-5 mg dayG . Manganese acts as a cofactor for several adult34. The Na is the most prominent cation in extracellular enzymes involved in metabolic processes necessary for the fluids, it is crucial in the maintenance of osmotic pressure of skeletal development, reproductive function and growth. This the body fluids and preserves normal function of the nervous element is also involved in urea formation, metabolism of and muscle36. A ratio of sodium ion to potassium ion less than amino acids, cholesterol and carbohydrates45. The zinc content one (Na+/K+<1) has been reported to be suitable for reducing of 4.40 mg/100 g is also relatively high and may be used to high blood pressure. It, therefore, suggested that the plant supplement up to 31.4% of RDA of 4-14 mg dayG1 in children could be a good source of food for hypertensive patients. The and adults, respectively46. The Zn is a vital micronutrient calcium content (485.00 mg/100 g) indicated that this plant required for the structural and functional integrity of biological can contribute meaningful amount of dietary calcium which membranes, maintaining homeostasis, regulation of insulin is needed for growth and maintenance of bones, teeth and production, regulation of glucose utilization by muscles and muscle and as such may be used as supplements in diets low fat cells and detoxification of free radicals47. Copper content of in calcium ion. Calcium acts as a vital second messenger in 1.55 mg/100 g was slightly above the RDA of 0.7-1.1 mg dayG1 blood coagulation, hormone secretion action, muscle in children and adults, respectively46. The Cu is involved in the contraction and nerve function37. proper functioning of key enzymes like cytochrome C oxidase, Phosphorus content of 400 mg/100 g obtained in this amine oxidase, catalase, peroxidase, ascorbic acid oxidase, study indicated that V. mespilifolia can contribute up to among others and plays a role in iron absorption. It is a 40% of RDA of the 200-1,000 mg dayG1 needed in children and necessary micronutrient for bone development, pigmentation, adults, respectively. Phosphorus is important in the synthesis hair growth, reproductive system, haematologic and of phospholipids and phospho-proteins38. It is also important neurologic systems48. for healthy bones and teeth. It is found in every cell and maintains normal cell growth and repairs. According to Shivraj Essential oil composition: The detailed chemical profile and Khobragade39, phosphorus maintains blood sugar level, of V. mespilifolia essential oil is given in Table 4. The acid-base balance and normal heart beat level. Though the SFME of 100 g of the plant yielded 0.46% of essential oil. magnesium content (140 mg/100 g) observed in this plant Total number of chemical constituents identified from the was low, it could still contribute about 31.1% out of the RDA essential oil was 15. The essential oil consisted mainly of of 450 mg dayG1 in human40. Magnesium acts as a cofactor to 2,5-dimethylhexa-2,4-diene (84.14%), "-gurjunene (5.33%), several enzymes (like kinases) which participate in energy and kaur-16-ene (2.15%), acetamide, N-(3-nitrophenyl)-2,2- protein production processes. It is also vital in strengthening dichloro (1.75%) and "-elemene (1.20%). The remaining cell membrane structure and modulates glucose transport 10 compounds accounted for less than 1% (Table 3). The across cell membranes41. In addition, it is very important in the various compounds present were grouped into monoterpenes formation and function of bones, muscles and prevents high ((-terpinene, 4-terpineol, 2, 3-epoxygeraniol), sesquiterpene blood pressure and depression. It also plays a crucial role in (2, 3-epoxygeraniol, "-elemene), diterpene (kaur-16-ene) muscle contraction, nerve transmission and boosting of the alkanes (9-octyleicosane, octadecane), apocarotenoids immune system39. The iron content 26.5 mg/100 g was (hexahydrofarnesyl acetone) and alkenes (2, 5-dimethylhexa-2, higher than the Recommended Daily Allowance (RDA) of 4-diene). 9-15 mg dayG1 in children and adults, respectively41, thus Essential oils are aromatic and volatile liquids, which are making the plant a good source of iron. Iron is an important characterized by a strong odour, rarely coloured, andhave element which aids in the transport of oxygen, electrons and adensity lower than that of water. They could be synthesized

65 Res. J. Bot., 12 (2): 38-45, 2017

Table 4: GC-MS profile of essential oils of Vernonia mespilifolia Retention time Name of compounds Area (%) 4.994 γ-terpinene 0.43 5.972 4-terpineol 0.60 7.529 2,6-dibromopyridine 0.82 8.253 "-gurjunene 5.33 9.048 "-elemene 1.20 9.448 Cyclohexyl(2,3-dimethylphenyl)methanol 0.66 9.661 2,5-dimethylhexa-2,4-diene 84.14 9.781 2,3-epoxygeraniol 0.80 9.895 Acetamide, N-(3-nitrophenyl)-2,2-dichloro 1.75 10.020 Cyclohexyl-(3,5-dimethylphenoxy)-dimethylsaline 0.41 10.084 Hexahydrofarnesyl acetone 0.52 10.469 2,10,10-trimethyltricyclo[7.1.1.0(2,7)]undec-7-en-6-one 0.37 11.308 Octadecane 0.37 11.399 Kaurene 2.15 12.195 9-octyleicosane 0.45 from all plant organs (flowers, buds, seeds, leaves, twigs, bark, SIGNIFICANCE STATEMENTS herbs, wood, fruits and root) and they are stored in secretory cells, epidermal cells or glandular trichomes49,50. Essential oils The present study gives insights into the essential oil and are known to only represent a small fraction of a plant’s nutritional compositions of Vernonia mespilifolia, a medicinal composition, despite their important roles such as food plant commonly used in South Africa. This study revealed that production, cosmetic and pharmaceutical industries51. this plant had high amount of certain important mineral Essential oils consist largely of small molecule whose main components when compared with some conventional constituents includes terpenes (oxygenated or not), with vegetables. It was also observed that the plant is rich in monoterpenes and sesquiterpenes. Nonetheless, allyl and carbohydrate which serves as a major source of energy and propenyl phenols (phenylpropanoids) are also important large amount of fibre which will help aid food digestion, components of some essential oils52. These substances are protein and low amount fat. Finally from our findings this responsible for the fragrance and for different biological plant species could be used to boast the immune system as a properties (anti-inflammatory, antimicrobial, anti-cancer, result of its rich essential oil compounds with therapeutic antiviral, anti-hyperglycemic and immunomodulatory importance, mineral and nutrient compositions and this could 53-55 activities . The essential oils of V. mespilifolia had not been be the reason of its therapeutic application in folkloric evaluated previously. However, the constituents as revealed medicine. in this study are very important. For instance, "-gurjunene has 56-58 been implicated to show insecticidal potential . According ACKNOWLEDGMENT to Tao et al.59 "-elemene is reported to have anti-cancer ability in both in vitro and in vivo models. Kaurene has been The authors would like to thank the Govan Mbeki reported to possess to the ability to induce apoptosis in Research Development Centre, University of Fort Hare, South 60-63 human leukemia cells and antibacterial activity . Acetamide, Africa for providing financial support. N-(3-nitrophenyl)-2,2-dichloro possess anticancer activity64. REFERENCES CONCLUSION

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CHAPTER FIVE

Phytochemical contents and antioxidant activities of Kedrostis africana, Vernonia mespilifolia and their combination

Chapter Five Phytochemical contents and antioxidant activities of Kedrostis africana, Vernonia mespilifolia and their combination

Contents Pages

Introduction ...... 71

Methodology ...... 72

Results and discussion ...... 79

Conclusion ...... 91

References ...... 92

INTRODUCTION Reactive oxygen species are normally produced during normal mitochondrial oxidative metabolism as well as in cellular response to xenobiotics and bacterial invasion oxidative (Ray et al. 2012). These reactive species such as singlet oxygen, superoxide ion, hydroxyl ion and hydrogen peroxide, are extremely reactive and toxic molecules which cause oxidative damage to important biomolecules such as nucleic acids, proteins, enzymes and lipids when the body cellular antioxidant defense system is overwhelmed thus causing a myriad of chronic and degenerative diseases in the body (Shukla et al. 2011). The use of natural sources of antioxidants from plants has attracted much interest as they have the capability of boosting the antioxidant capacity of the plasma, thus reducing the onset of certain disorders such as cancer, diabetes, obesity, cardiovascular and neurodegenerative diseases. The plant kingdom has for long been an indispensable source of therapeutic agents and many modern drugs are semi- synthesized or isolated from these natural sources (Rischer et al. 2013). The leaves, flowers, stems, roots, seeds, fruit and bark are the major parts of plants where most phytochemical components that produce certain physiological action in the human body are found (Afify et al. 2012). In recent times, the amount of polyphenols in plants and antioxidant activities depends on biological factors such as (genotypes /organ used) and environmental conditions like water stress, temperature and light intensity (Bano et al. 2003). Also, the amount of phenolic compounds extracted from plant materials depends on the polarity of the solvent used used for extraction (Djeridane et al. 2006).

From literature, some medicinal plants have been shown to possess mixtures of phytochemical compounds such as flavonoids, tannins, and polyphenols which inhibit or reduce oxidative deterioration of lipids, proteins, and DNA, consequently preventing neurodegenerative

diseases, atherosclerosis, chronic inflammatory diseases, carcinogenesis, and other pathological disorders (Karbach et al. 2014; Turati et al. 2015).

V. mespilifolia is used in folk and traditional medicine for the management of weight loss, hypertension in humans (Mbaebie and Afolayan, 2010) and heart water disease in goats (Dold and Cock, 2001); while K. africana tuber is used in Khoi-San and Cape Dutch medicine as an emetic, purgative, diuretic and treatment of dropsy (van Wyk, 2008). The crushed fresh bulb is used ethnomedically for the management of obesity in Eastern Cape, South Africa (George and Nimmi, 2011) and combination of both plants are used in ethnomedicine for the management of obesity (Mbaebie and Afolayan, 2010). To date, no studies have investigated the free radical scavenging or antioxidant activities of Vernonia mespilifolia and Kedrostis africana and the combination of both plants. This study investigated the preliminary screening of extracts of the plants for their phytochemical composition and antioxidant activities in order to understand their potential for therapeutic use.

METHODOLOGY Location and collection of sample

The whole plant parts of Vernonia mespilifolia and tubers of Kedrostis africana were collected in June 2015 from Zihlahleni Village and Fort Beaufort area respectively both in Eastern

Cape, South Africa. These plants were authenticated by Mr. Tony Dold of Selmar Schonland

Herbarium, Rhodes University, South Africa, and a voucher specimen (Unuofin Med,

2015/1&2) was prepared and deposited at the Giffen Herbarium, University of Fort Hare.

Preparation of extracts

The whole plant part of V. mespilifolia and tubers of K. africana were rinsed with deionised water and gently blotted with a paper towel to remove excess water and subsequently oven-

dried (LABOTEC, South Africa) at 40°C for 72 hours until constant weight was achieved. The dried sample were then ground into powder (Polymix® PX-MFC 90D Switzerland) and stored at 4°C till needed for analyses. Equal proportions of the ground powder of both plants (1:1) were mixed together to form a combination. The ground sample (200 g) was weighed into 3 separate conical flasks containing (2 L) acetone, ethanol, and water respectively, and shaken on an orbital shaker (Orbital Incubator Shaker, Gallenkamp) for 48 hours. The crude extracts were filtered under pressure using a Buchner funnel and Whatman No. 1 filter paper. The acetone and ethanol extracts were further concentrated to dryness to remove the solvents under reduced pressure using a rotary evaporator (Strike 202 Steroglass, Italy), while the aqueous filtrate obtained was concentrated using a freeze dryer (Vir Tis benchtop K, Vir Tis Co.,

Gardiner, NY). The acetone, aqueous and ethanol extracts were stored at 4oC.

Reagents and chemicals used

Solvents and chemicals used were purchased from Merck and Sigma-Aldrich, Gauteng, South

Africa. These include Folin-Ciocalteu reagent, anhydrous sodium carbonate (Na2CO3), aluminium trichloride (AlCl3), sodium nitrite (NaNO2), sodium chloride, 2,2 diphenyl-1- picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), vanillin, aluminum chloride (AlCl3), potassium acetate (CH3CO2K), ferric chloride (FeCl2),

BHT, ascorbic acid, rutin, n-butanol, diethyl ether, ammonia solution, acetone, ethanol, hydrochloric acid, sodium hydroxide, phosphate buffer, potassium ferricyanide [K3Fe(CN)6], ammonium molybdate, sodium phosphate, trichloroacetic acid (TCA), glacial acetic acid

(CH3COOH), sodium nitroprusside (Na2[Fe(CN)5NO]2H2O). All the reagents used in this study were of analytical grade.

In-vitro quantitative phytochemical evaluation

Total Phenolic

Phenol content in the plant extract was estimated spectrophotometrically using the Folin-

Ciocalteu’s reagent method as described by Sen et al. (2013) with some modifications. 0.5 mL of the plant extracts (1 mg/ml) and standard gallic acid (0.02 mg/ mL to 0.1 mg/mL) was dispensed in different test tubes. To this, 2.5 mL of 10% (v/v) Folin-Ciocalteu’s reagent was added and the mixture was vortexed. The reaction was allowed to stand at room temperature for about 5 mins, after which 2 mL of 7.5% (w/v) anhydrous sodium carbonate was added to the solution, vortexed and incubated at 40oC for 30 mins. In the control tube, the extract volume was replaced by methanol. After incubation, the absorbance was measured at 765 nm using a

UV- 3000 PC spectrophotometer. The experiment was done in triplicate. The phenol content was extrapolated from the gallic acid standard/calibration graph equation; y = 8.7668 x +

0.1977, R2 =0.9983, and calculated using the following formula:

C=c×V/ m, where

C= total content of phenolic compounds in mg/g plant extract in GAE or mg GAE/g extract c= the concentration of gallic acid established from the calibration curve in mg/ml

V= the volume of extract in ml; m= the weight of extract used in the assay in g

Flavonoid determination

The colorimetric Aluminum chloride assay was used to determine the flavonoid content in the plant extract according to the method described by Ghasemzadeh et al. (2014) with little modification. This method is based on the quantification of the yellow-orange colour produced by the interaction of flavonoid with AlCl3. Briefly 0.5 mL (1 mg/mL) aliquots of the different

solvent extracts and different concentrations (0.2 to 1 mg/mL) of quercetin standard were dispensed in different test tubes, 2 mL of distilled water and 0.15 mL of 5% sodium nitrite was added to the test tubes and the mixture was allowed to stand for 6 mins. Thereafter, 0.15 mL of 10% AlCl3 was added to the solution, allowed to stand for another 5 mins followed by the addition of 1mL of 1 M sodium hydroxide. The solution was made up to 5 mL with distilled water and the absorbance measured using a spectrometer at 420 nm. A control solution containing 0.5 mL of distilled water instead of the extract/standard was used as blank. The experiment was done in triplicate. The flavonoid content was calculated using the calibration curve equation, y = 1.1734 x + 0.1543 R² = 0.9698. and expressed as mg of quercetin equivalent

(QE)/g using the formula CV/m in the same manner as described in the phenolics above.

Proanthocyanidin determination

Determination of proanthocyanidin was as described by Falleh et al. (2009). The reaction mixture contained 0.5 mL of 1 mg/mL of the extract solution of standard catechin at different concentrations (0.02 mg/mL to 1 mg/mL) plus 3 mL of vanillin-methanol (4% w/v) and 1.5 mL of hydrochloric acid. The mixture was vortexed and allowed to stand for 15 min at room temperature, while a control solution which had neither an extract nor catechin was used as blank. The absorbance was measured at 500 nm using a UV- 3000 PC spectrophotometer. The experiment was done in triplicate. Proanthocyanidin content was calculated using the calibration curve equation: y = 0.9038 x + 0.0449, R2 0.9951 and expressed as mg catechin equivalent (CE)/g using the formula, CV/m as earlier described for in phenol.

Determination of Tannins

Tannin was determined as described previously Mbaebie et al. (2012). Briefly, 0.2 g of plant extract was added to 20 mL of 50% methanol. This was mixed thoroughly and placed in a water

bath at 80∘C for 60 min. The extract was filtered into a 100 mL volumetric flask; 20 mL of distilled water was added, followed by 2.5 mL of Folin-Ciocalteu reagent and 10 mL of 17%

Na2CO3.This was thoroughly mixed together and made up to 100 mL using distilled water. The mixture was allowed to stand for 20 min until a bluish-green color developed. The different tannic acid standard solutions concentrations used ranged from 0 to 10 ppm. The absorbance of the tannic acid standard solutions and plant extracts were measured after color development at 760 nm using UV- 3000 PC spectrophotometer.The results were expressed as mg/g of tannic acid equivalent using the calibration curve: Y = 154.45x; R2 = 0.9585.

In-vitro anti-oxidant analyses

The antioxidant capacities of the different extracts were measured using DPPH radical scavenging activity, ABTS radical scavenging activity, hydrogen peroxide and nitric oxide scavenging activities. These measurements were made against standard antioxidants, including

BHT and Rutin.

ABTS (2, 2’-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid) radical scavenging activity The method described by Asowata-Ayodele et al. (2016) was adopted for the determination of

ABTS activity of the plant extract. Briefly, the working solution was prepared by mixing equal volumes of two stock solutions of 7 mM ABTS and 2.45 mM potassium persulfate which was in equal volumes (1:1) and allowed to react for 12 h at room temperature in the dark to release

ABTS radicals (ABTS+). The resultant green-coloured solution was further diluted by mixing

1 mL of the ABTS+ solution with about 50 mL of methanol to obtain an absorbance of 0.700

± 0.006 at 734 nm. Upon obtaining the desired absorbance, 1 mL of the resultant solution is then mixed with 1 mL of the plant extract/or standards at different concentrations (0.005 mg/mL to 0.08 mg/mL). After 7 min, the reduction in absorbance is measured at 734 nm using a spectrophotometer. The percentage inhibition of ABTS+ by the extract or standard was

calculated from the following equation: % inhibition = [(Abs control – Abs sample)] / (Abs control)] × 100

DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity assay The method described by Asowata-Ayodele et al. (2016) was used for the determination of

DPPH free radical scavenging activity. Briefly, a solution of 0.135 mM DPPH radical in methanol was prepared. A 1 mL of this solution was mixed with 1 mL (0.005 mg/mL to 0.08 mg/mL) each of the plant fractions/standards drugs (BHT, Rutin) at different concentrations.

The reaction mixture was then vortexed thoroughly and left in the dark at room temperature for 30 min. The absorbance of the mixture was measured spectrophotometrically at 517 nm.

The actual decrease in absorbance was measured against that of the control. The scavenging ability of the plant extract was then calculated using the equation:

DPPH Scavenging activity (%) = [(Abs control – Abs sample)/ (Abs control)] ×100;

Where; Abs control is the absorbance of DPPH + methanol; Abs sample is the absorbance of

DPPH radical + sample/or standard.

Hydrogen Peroxide (H2O2) Scavenging Activity.

The H2O2 inhibitory activity of the extracts was assessed by the method of Asowata-Ayodele et al. (2016). Briefly, a solution of 4 mM H2O2 was prepared in phosphate buffer (0.1 M; pH

7.4) and incubated for 10 min. One milliliter of each plant extract at different concentration

(0.025 to 0.4 mg/mL) was added to 0.6 mL of hydrogen peroxide solution.The absorbance of the hydrogen peroxide at 230 nm was determined after 10 min against a blank solution containing phosphate buffer solution without hydrogen peroxide. The positive controls used were BHT and vitamin C. The percentage scavenging of hydrogen peroxide of samples was

calculated using the formula: H2O2 inhibition capacity (%) = [1 − (H2O2 abs of sample / H2O2 abs of blank )] × 100.

Nitric oxide scavenging activity A 2 mL aliquot of 10 mM sodium nitroprusside prepared in 0.5 mM phosphate buffer saline

(pH 7.4) was mixed with 0.5 ml of plant fractions, vit C and BHT individually at different concentrations (0.025 to 0.4mg/mL). The mixture was incubated at 25°C for 150 min. 0.5 mL of the incubated solution was mixed with 0.5 mL of Griess reagent [1 mL sulfanilic acid reagent

(0.33% prepared in 20% glacial acetic acid) and 1 mL of naphthalene diamine dichloride (0.1% w/v) at room temperature for 5 min]. The mixture was incubated at room temperature for 30 min, followed by the measurement of the absorbance at 540 nm. A solution containing water instead of the extract/standard was used as a control. The amount of nitric oxide radicals inhibited by the extract was calculated using the equation:

NO radical scavenging activity (%) = [(Abs control – Abs sample)]/(Abs control)] × 100, where

Abs control is the absorbance of NO radicals + methanol and Abs sample is the absorbance of

NO radical + extract or standard.

Statistical analysis

All data were expressed as mean ± standard deviation (SD) of three replications. Statistical analysis was performed by ANOVA. Where the data showed significance (p < 0.05), a mean separation was done using the Fischer’s LSD with the aid of MINITAB 17 statistical package.

RESULTS AND DISCUSSION In recent days, people attempted to reduce a hazard or handle a specific health situation through enhanced food diet. Plants and fruits have evolved diverse phytochemicals, which have a high amount of antioxidant potential. Naturally derived antioxidants are multifunctional and have increasingly gained interest as alternatives to synthetic antioxidants in the process of oxidation in complex food systems (Wang et al. 2009). Phytochemical analysis performed on these plant extracts revealed the presence of constituents, with renowned therapeutic and physiological activities (Sofowora, 1993). Numerous studies have reported the antioxidant properties of phenolic compounds present in diverse parts of various medicinal plants (Krings & Berger

2001; Wang et al. 2003). Phenolics, have received great attention because of their antioxidant properties and they can potentially interact with biological systems and play an important role in anticancer, anti-inflammatory, and antimicrobial activity (Wang et al. 2003; Abu-Reidah et al. 2013). Phenolic compounds, such as terpenoids and flavonoids, are the principal antioxidants that exert a scavenging effect on free radicals and reactive oxygen species

(Hossain et al. 2011). Flavonoids possess numerous mechanism at which they scavenge free radical. Examples of such mechanism are chelation of metal ions, such as iron and copper; the scavenging of free radicals; and the inhibition of enzymes responsible for free radical generation (Benavente et al. 1997; Agati et al. 2012).

In this study, total flavonoid, phenolic, proanthocyanidins, and tannin concentrations, as well as the antioxidant activities of different solvents extracts were determined in V. mespilifolia,

K. africana and combination of both plants. These parameters usually depend on the type and polarity of solvent (Daoud et al. 2015; Sharifi-Rad et al. 2015). As shown in Table 1, acetone extracts of the combination of both plants (AC) gave the highest concentration of polyphenol

(144.59 mg GAE/g) and flavonoids (803.93 mg QE/g) while the acetone extract of K. africana gave the highest concentration (585.64 mg CE/g) of proanthocyanidins. The ethanol extract of 79

the combination of both plants (EC) (1.245 mg TAE/g) exhibited the highest concentration of tannins as shown in Table 1. This is similar to the results of Sharifi-Rad et al. (2015) and

Asowata-Ayodele et al. (2016) obtained using Chrozaphora tinctoria and Lippia javanica respectively where the acetone extract gave the highest yield of total polyphenols, flavonoids and tannins. In recent times, various scientists have been appraising the effects of extraction solvents on natural products. The polarity of solvent used for extraction have a great impact on the yield of different phytochemicals present in plants; and according to Eloff, (1998) and

Daoud et al. (2015), acetone extract contains a better-quality of extracted bioactive components and antimicrobial substances.

The highest antioxidant activity in plants is attributed to polyphenols among other secondary metabolites (Farasat et al. 2014). Phenolics have the ability of oxidizing a broad spectra of free radicals to their stable radical intermediates (Koolen et al. 2013). They also serve as electron donors, metal chelators, singlet and triplet oxygen quenchers (Tshivhandekano et al. 2014).

Tannins bind and precipitate microbial proteins thus making bacteria malnourished, and are also used in small concentration for digestion complications because of the tendency to bind to iron and making it less available for heme formation (Babaa and Malik, 2015).

The result obtained from the phytochemical analysis of the combination of both plants V. mespilifolia (VM) and K. africana (KA) revealed that the combination of both plants (C) had greater polyphenol, flavonoid and tannin contents. This could be the reason why the combination of both is believed to be more effective in the management of weight loss

(Afolayan and Mbaebie, 2010), except for the acetone extract of K. africana which had the greater concentration of proanthocyanidins.

Table 5. 1. Polyphenolic contents of various solvent extracts of V. mespilifolia, K. africana and their combination. Extract Polyphenol Proanthocyanidins Flavonoids Tannins (mg

(mg GAE/g) (mg CE/g) (mg QE/g) TAE/g)

Acetone combined 144.59 ± 0.02a 316.85 ± 0.01b 803.93 ± 0.01a 1.092 ± 0.01b

Acetone V. mespilifolia 99.04 ± 0.00d 132.11 ± 0.00d 542.87 ± 0.01b 0.984 ± 0.00c

Acetone K. africana 10.51 ± 0.01g 585.64 ± 0.02a 529.23 ± 0.01c 0.495 ± 0.01e

Aqueous combined 14.46 ± 0.03f 7.71 ± 0.02i 78.97 ± 0.01h 0.269 ± 0.01h

Aqueous V. mespilifolia 39.07 ± 0.01e 34.56 ± 0.00g 171.58 ± 0.02g 0.36 ± 0.01af

Aqueous K. africana 5.32 ± 0.00i 15.04 ± 0.01h 46.61 ± 0.02i 0.301 ± 0.02g

Ethanol combined 109 ± 0.00b 106.33 ± 0.02e 496.28 ± 0.00d 1.245 ± 0.01a

Ethanol V.mespilifolia 106.99 ± 0.01c 83.9 ± 0.00f 464.75 ± 0.02e 0.932 ± 0.02d

Ethanol K. africana 5.98 ± 0.01h 222.91 ± 0.01c 285.78 ± 0.01f 0.937 ± 0.00d

Values are expressed as mean ± standard deviation of three replicates; mg GAE/g = milligram Gallic acid equivalent per gram of extract; mg QE/g = milligram quercetin equivalent per gram of extract; mg TAE/g = milligram tannic acid equivalent per gram of extract; mg CE/g = milligram catechin equivalent per gram of extract.

In-vitro antioxidant activities DPPH free radical scavenging assay Figure 5.1 shows the DPPH free radical scavenging activity of the various extracts of V. mespilifolia, K. africana and combination of both plants. The scavenging ability of the various extracts was in a concentration dependent manner and in the following order ethanol V. mespilifolia > ethanol combined > acetone V. mespilifolia > acetone combined > acetone K. africana > aqueous K. africana > ethanol K. africana > aqueous V. mespilifolia > aqueous combined. The scavenging ability of the various extracts was in a concentration dependent manner. The IC50 values for the various extracts shown in Table 5.2 reveals that ethanol V. mespilifolia, ethanol combined, acetone V. mespilifolia and acetone combined showed greater scavenging activity on DPPH than BHT (IC50 = 0.1549 mg/mL) one of the standards. The ethanol extract of V. mespilifolia showed maximum scavenging activity among all the extracts used. It has been reported that the antioxidant activity of plant extracts containing polyphenol components possess abilities to be donors of hydrogen atoms or electrons and to capture the free radicals (Shon et al. 2003). This is evident from the DPPH scavenging ability of ethanol

V. mespilifolia, ethanol combined, acetone V. mespilifolia and acetone combined which had high phenolic contents. According to Chung et al. (2006), DPPH radical-scavenging assay is one of the widely used methods of evaluate the scavenging potentials of plant extracts (Chung et al. 2006).

DPPH free radical scavenging assay A

100 e e c d e 80 e d a 60 d c d d a 40 c

c c a activity(%) scavenging a 20 a b a b DPPH radicalDPPH b b b 0 0,005 0,01 0,02 0,04 0,08

Acetone extract Aqueous extract Ethanol extract BHT Rutin

B 100 d c d c 80 d c

60 d b c 40 c (%) 20 a b b a ab b a b b a a a a b a

0 DPPH radicalDPPH

0,005 0,01 0,02 0,04 0,08 scavenging activity scavenging Acetone extract Aqueous extract Ethanol extract BHT Rutin

100 e e e d d 80 d c 60 d e a c 40 c a d a c 20 a c a a DPPH radical DPPH b b b b b 0 scavenging activity (%) activity scavenging 0,005 0,01 0,02 0,04 0,08 Acetone extract Aqueous extract Ethanol extract BHT Rutin

ABTS radical scavenging activity The ABTS radical scavenging activities of V. mespilifolia, K. africana and combination of both plants are shown in Figure 5.2. The activity was concentration dependent and the maximum scavenging activity was found in the ethanolic extract of V. mespilifolia (IC50 = 0.0119 mg/mL), which was higher than that of rutin standard (IC50 = 0.992 mg/mL), followed by acetone extract (IC50 = 0.0134 mg/mL). In K. africana, ethanol extract showed greater scavenging activity than the acetone extract. A synergistic effect on ABTS radical scavenging was observed with the combination K. africana and V. mespilifolia in a ratio of 1:1. The IC50 value of the combined plants exhibited highest scavenging ability in the ethanol extract 0.0134 mg/mL which is equal to that of the acetone extract of V. mespilifolia (Table 5.2). It was observed that the acetone, aqueous and ethanol extracts of K. africana showed 4, 2 and 1.1- fold reduction in IC50 value on synergism with V. mespilifolia, while aqueous and ethanol extracts of V. mespilifolia had a 0.44 and 0.89-fold increase in IC50 respectively. Also the results on ABTS scavenging activity are in well agreement with amount of phenolic constituents present in respective extract. Phenolics present in V. mespilifolia extracts are able to terminate radical chain reaction by converting free radicals to more stable products in greater extent, thus showing more activity as compared to other extracts.

ABTS radical scavenging activity A

120 c a a 100 a c a c d d d d e b 80 c c 60 c a b 40 a a a a b 20 b b

ABTS radical ABTS 0

0,005 0,01 0,02 0,04 0,08 scavenging activity(%)scavenging

Acetone extract Aqueous extract Ethanol extract BHT Rutin

B 150

e d d 100 e d e e e d c d a a b 50 a c c

c b activity(%) scavenging a c a b b ABTS radical ABTS 0 b 0,005 0,01 0,02 0,04 0,08 Acetone extract Aqueous extract Ethanol extract BHT Rutin

120 a 100 e d d e e e d c e c 80 a d 60 d c b (%) 40 c a c a 20 a b b ABTS radical ABTS b b

scavenging activity scavenging 0 0,005 0,01 0,02 0,04 0,08

Acetone extract Aqueous extract Ethanol extract BHT Rutin

Figure 5. 2: ABTS radical scavenging activity of different extracts from A) V. mespilifolia, B) K. africana, and C) combination of both plants at different concentrations. Each value represents mean ± SD (n = 3)

Nitric oxide radical scavenging activity

The IC50 for the nitric oxide radical scavenging activities of V. mespilifolia, K. africana and combination of both plants activities are shown in Table 5.2. The activity was concentration dependent as shown in Figure 5.3. The maximum scavenging activity was found in the ethanolic extract of V. mespilifolia (IC50 = 0.0003 mg/mL), which was higher than that of rutin and BHT standards (IC50 = 0.0768 and 0.0294 mg/mL) followed by acetone extract of V. mespilifolia (IC50 = 0.0134 mg/mL). In K. africana, the ethanol extract showed greater scavenging activity than acetone extract. The ethanol extract of the combined plants gave the highest scavenging of nitric oxide radicals IC50 0.3071 mg/mL. The acetone extract of K. africana showed a 2 -fold reduction in IC50 value on synergism with V. mespilifolia, while acetone and ethanol extracts of V. mespilifolia had a 2.6 and 1024-fold increase in IC50 respectively. Nitric oxide (NO) is a reactive free radical produced by phagocytes and endothelial cells, to yield more reactive species such as peroxynitrite which further decompose forming OH radical. V. mespilifolia, K. africana and combination of both plants could significantly reduce the level of nitric oxide which is reported to play a crucial role in inflammation (Moncada et al. 1997). Also, This results are very promising because this suggests that combining the two plants will reduce the concentration or amount needed for treating various diseases and disorders (Afolayan and Mbaebie, 2010).

Nitric oxide radical scavenging activity A

80 c c c d c c d a a 60 d d e a a d e a a a b b 40 a b b 20 b

0 oxide radical (%) radical oxide Inhibition ofnitric Inhibition 0,025 0,05 0,1 0,2 0,4 Acetone extract Aqueous extract Ethanol extract BHT Rutin

80 d d 60 d d b e b c e e b c e c d c a e 40 c b a b a 20 a

oxide radical (%) radical oxide a Inhibition ofnitric Inhibition 0 0,025 0,05 0,1 0,2 0,4 Acetone extract Aqueous extract Ethanol extract BHT Rutin

C 80 c 60 d d d e b d e e b a d b a c a 40 e b a c a c 20 c a b

0 oxide radical (%) radical oxide

Inhibition ofnitric Inhibition 0,025 0,05 0,1 0,2 0,4 Acetone extract Aqueous extract Ethanol extract BHT Rutin

Figure 5. 3: Nitric oxide radical scavenging activity of different extracts from a) V. mespilifolia, b) K. africana, and c) combination of both plants at different concentrations. Each value represents mean ± SD (n = 3).

Hydrogen peroxide radical scavenging activity

Results of the hydrogen peroxide scavenging activity of V. mespilifolia and K. africana and their combination is as shown in Figure 5.4. The IC50 value (Table 5.2) of the acetone, aqueous and ethanol extracts of V. mespilifolia were 0.1109, 0.0249 and 0.0865 mg/mL respectively.

These extracts appeared to be better scavengers of hydrogen peroxide radicals than standard rutin (IC50 = 0.1172 mg/mL). The acetone and ethanol extracts of K. africana showed moderate hydrogen peroxide scavenging potentials with (IC50 = 0.1262 and 0.0562 mg/mL respectively) while the aqueous extract showed poor hydrogen peroxide scavenging potential (IC50 = 1.7873 mg/mL), but the combination of both plants exhibited higher scavenging activity in acetone, aqueous and ethanol extracts when compared to the activity of each individually. The aqueous extract of the combined plants had the best scavenging potential when compared with the other the extracts and standards used.

Hydrogen peroxide radical scavenging activity a)

100 d d d d d b c b b c a e 80 a b c e 60 a e b 40 c a a 20 peroxide (%) peroxide a c e 0

Inhibition ofhydrogen Inhibition 0,025 0,05 0,1 0,2 0,4 Acetone extract Aqueous extract Ethanol extract BHT Rutin b)

100 b b b b b b a b d d c 80 c a a c a a 60 c a d 40 c e 20 a a e peroxide (%) peroxide 0

0,025 0,05 0,1 0,2 0,4 Inhibition ofhydrogen Inhibition Acetone extract Aqueous extract Ethanol extract BHT Rutin c)

100 d d d b c d b b a a 80 b a d a c e b 60 c c a 40 e 20 a c

peroxide (%) peroxide c c 0

Inhibition ofhydrogen Inhibition 0,025 0,05 0,1 0,2 0,4 Acetone extract Aqueous extract Ethanol extract BHT Rutin

Figure 5. 4: Inhibition of hydrogen peroxide activity of different extracts from a) V. mespilifolia, b) K. africana, and c) combination of both plants at different concentrations. Each value represents mean ± SD (n = 3). 89

Table 5. 2. IC50 values of the solvent of V. mespilifolia, K. africana and their combination and standard drugs

Extract/Standard IC50 (mg/mL)

DPPH ABTS Nitric oxide H2O2

Acetone combined 0.0779 0.0304 0.3025 0.0715

Acetone V. mespilifolia 0.0587 0.0134 0.1159 0.1109

Acetone K. africana 0.3031 0.0649 0.6204 0.1262

Ethanol combined 0.0553 0.0134 0.3071 0.1182

Ethanol V.mespilifolia 0.0392 0.0119 0.0003 0.0865

Ethanol K. africana 0.3978 0.0544 0.1514 0.0568

Aqueous combined 1.0962 0.0890 0.1690 0.0033

Aqueous V. mespilifolia 1.8721 0.0392 0.6034 0.0249

Aqueous K. africana **** 0.0977 0.1357 1.7873

BHT 0.1549 0.0036 0.0294 ****

Rutin 0.0029 0.9962 0.0768 0.1172

IC50 = concentration of extract or standard producing 50% antioxidant activity; ***Values too high and not relevant. Note that a low IC50 value indicates a high antioxidant activity of a compound.

CONCLUSION This study revealed the effect of different solvents on the extraction of polyphenols and antioxidant activities of V. mespilifolia and K. africana and their combination. Results revealed that the acetone extract of the combined plants exhibited the highest concentration of total phenols and flavonoids while the ethanol extract of the combined plants gave the highest tannin content; and the acetone extract of K. africana exhibited the highest proanthocyanidin content.

In addition, the ethanol extracts of V. mespilifolia gave the best scavenging activity for DPPH,

ABTS radical activity, and nitric oxide radicals while the aqueous extract of the combination of both plants exhibited the best scavenging ability against hydrogen peroxide. In all, these plants extracts strong scavenging ability could be employing in combating conditions induced by oxidative stress hence, expanding their application singly and in combination as a therapeutic agent is recommended.

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CHAPTER SIX

Evaluation of the anti-microbial activities of Kedrostis africana, Vernonia mespilifolia and their combination on microbes associated with obesity

Chapter Six Evaluation of the anti-microbial activities of Kedrostis africana, Vernonia mespilifolia and the combination of both plants on microbes associated with obesity

Contents Pages

Introduction ...... 101

Materials and methods ...... 102

Results ...... 105

Discussion ...... 108

Conclusion ...... Error! Bookmark not defined.

References ...... 111

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INTRODUCTION The role of the gut microbiota in the pathogenesis of obesity has emerged into an important research area in the etiology and management of obesity (Backhed et al. 2004). Gram-negative opportunistic pathogens in the gut may be pivotal in the etiology obesity (Zhang et al. 2010;

2012). In the past decades, interest in the metabolic role of the communal gut microbes in humans focused on their potential to ferment indigestible nutrients, produce micronutrients, and reduce harmful toxins. However, it has become increasingly appreciated that host/microbe interactions help balance host vital functions and participate in health maintenance. The digestive microbiota is a complex ecosystem that consists of viruses, bacteria, fungi and parasites. Specific enterotypes have been identified and linked to diet and their antibiotic- mediated modulation can impact the metabolic profile of the host.

The human gut is one of the most compact populated ecosystem that has been explored (Siezen and Kleerebezem, 2011). The microbes in the human gut have been implicated in the etiology of myriad of diseases. As far back as 2005, the gut microbiome has received attention as a new focus point for the onset of obesity with a number of studies attesting to these findings (Ley et al. 2005, 2006; Turnbaugh et al. 2006). This has brought about the exploration of the structural and functional composition of obesity-related gut microbiome. The diversities in intestinal microflora composition in humans is suggested to be a key factor affecting energy homeostasis

(Ley et al. 2005). The two most prominent bacterial phyla in the gut of mice and humans are the Gram-negative Bacteroidetes and the Gram-positive Firmicutes (Ley et al. 2005; Eckburg et al. 2005). These microflora composition is strongly affected by dietary patterns. A high-fat and high-sugar Western diet increases the relative abundance of Firmicutes at the expense of the Bacteroidetes in animal models (Cotillard et al. 2013), whereas low-calorie diet induced weight loss may increase the relative abundance of Bacteroidetes in obese individuals (Ley et al. 2006). A particular configuration in the gut microbiota has being implicated in disease 101

which is often called bacterial “dysbiosis”. The ever growing report on the associations obesity and dysbiosis suggests that it may be useful to modulate the microbiome with targeted therapeutics and to restore it to a healthy or obese state (Ianiro et al. 2014).

Some of the fungal isolates associated with obesity are Candida albicans, Penicillium chrysogenum, Trichophyton tonsurans and Microsporum gypseum. Candida albicans and

Penicillium chrysogenum are also associated with oral cavity infections (Drozdowska and

Drzewoski, 2008). Trichophyton mucoides cause skin and athlete’s foot (Chin-Hong, 2006) while Microsporum gypseum is associated with gastro-intestinal, pulmonary and central nervous system infections (Eckburg et al. 2005). Multidrug resistance in microbial pathogens is an ongoing global problem. Thus, there is an urgent and constant need for exploration and development of cheaper, effective, new plant-based drugs with better bioactive potential and fewer possible side effects. However, there is a dearth of information on the evaluation of the antimicrobial potential of V. mespilifolia, K. africana and the combination of both plants, hence this current study is aimed at evaluating the anti-bacterial and fungal activities of V. mespilifolia, K. africana and the combination of both plants on different gut microbes associated with obesity using the agar dilution techniques.

MATERIALS AND METHODS Collection and extraction of samples were carried out as previously described in chapter five.

Rationale for the selection of the microorganisms

The bacteria and fungi used for this work were selected based on their association with the pathogenesis of obesity (Vajro et al. 2013)

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Microbial strains The selected bacterial strains for this study were three gram positive strains; Actinomyces odontolyticus (ATCC 17929), Lactobacillus sakei (ATCC 15521), Staphylococcus aureus

(ATCC 18824) and three gram negative Enterobacter cloacae (ATCC 13047), Pseudomonas aeruginosa (ATCC 19582) and Bacteriodes thetaiotomicron (ATCC 29741) strains. The fungal strains used for this investigation were Candida albicans (ATCC 10231), Microsporium gypsum (ATCC 24102), Penicillium chrysogenum (ATCC 10106), Trichophyton tonsurans

(ATCC 28942). All the microbes were obtained from the Microbiology Unit of the Medicinal

Plants and Economic Development Research Centre, Department of Botany, University of Fort

Hare.

Preparation of bacterial inoculum Direct colony suspension method was used in preparing the inoculum. Three to five morphologically similar colonies from fresh Muller Hinton agar plates were transferred with a loop into 5 mL of normal saline in a capped test tube and vortexed. The suspension formed was adjusted to give a turbidity equivalent to that of 0.5 McFarland standard to give an approximate

1.5 × 108 CFU/mL. The adjusted colony was then diluted to a ratio of 1:100 in Muller Hinton broth to give a colony suspension of 1 × 106 CFU/mL. Final suspensions of 1 × 104 CFU/spot was used for the agar dilutions.

Preparation of fungal inoculum Fungal strains were freshly sub-cultured on sterile Sabouraud Dextrose agar and incubated at

30oC for 4 days. The resultant cells and spores were washed into sterile normal saline and the eturbidity adjusted to 0.5 McFarland standard equivalent which resulted in a 1 × 106 CFU/mL.

The suspension was further diluted to a 1:10 ratio in Sabouraud Dextrose broth to give a turbidity of 5 × 105 CFU/mL.

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Dilution assays Agar dilution and broth microdilution assays as described by Wiegand et al (2008) and the

European Committee for Antimicrobial Susceptibility Testing (EUCAST, 2003) which are modifications from the guidelines of the Clinical and Laboratory Standard Institute (CLSI), were used for this study.

Preparation of extract

A stock solution of 100 mg/mL prepared in little amount of DMSO and made up with either

Muller Hinton or Sabouraud Dextrose Broth for anti-bacterial and fungal respectively was prepared. Two folds serial dilutions of the extract (50, 25, 12.5, 6.25, 3.125, 1.5625 mg/mL) were then prepared. Standard antiboiotics (Ciprofloxacin and Nystatin for bacterial and fungi respectively) were also prepared by 2-folds serial dilutions as described by Clinical and

Laboratory Standard Institute (CLSI).

Agar dilution assay Muller Hinton and Sabouraud Dextrose Agar were separately prepared according to the manufacturer’s description for antibacterial and antifungi screening respectively. The agar was autoclaved at 121o C for 15 min and allowed to cool to 50oC in a water bath. One millilitre from the 2-fold serial dilutions were added to the molten agar (19 mL) in the water bath, swirled and poured into petri dishes, allowed to cool and solidify. Ten microlitre each from both the prepared bacterial and fungal inoculum were delivered on to the solidified agar surface to give the desired final inoculum of 1 × 104 CFU/spot and 1 × 103 CFU/mL respectively. The concentrations of the extracts ranged from 0.1563 - 5 mg/mL. The concentration of

Ciprofloxacin (antibacterial standard) ranged from 2 - 64 µg/mL while Nystatin (antifungal standard) ranged from 0.5 - 16 µg/mL. Agar plates for bacteria were incubated at 37oC and readings were taken between 16-24 hrs of incubation; while for the fungi plates were incubated

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at 30oC and readings were taken after 36 - 48 hrs. The MICs were determined as the lowest concentration of extract inhibiting the visible growth of each organism on the agar plate.

RESULTS Antibacterial assay

The results of the antibacterial minimum inhibitory concentration (MIC) of the extracts against the tested bacterial strains are shown in Table 6.1. The result revealed that both gram(s) positive

(+ve) and negative (-ve) bacteria tested were susceptible to the crude extracts of V. mespilifolia,

K. africana and their combination, with the gram-negative bacterial strains been more susceptible to the crude extracts. Acetone extract of V. mespilifolia exhibited the highest activity (2.5 mg/mL) against B. thetaiotomicron while the acetone, aqueous and ethanol extracts of K. africana did not show any activity against this organism. In addition, B. thetaiotomicron was also resistant to the aqueous extracts of V. mespilifolia and their combination. A. odontolyticus, was more susceptible to both the acetone extracts of the combined plants and V. mespilifolia at MIC 2.5 mg/mL. However, A. odontolyticus was resistant to the aqueous extracts of both plants and combination tested and also to the acetone extract of K. africana. L. sakei was resistant to all the aqueous extracts tested, as well as the acetone extract of K. africana, but was susceptible to the acetone extract of V. mespilifolia with an MIC of 2.5 mg/mL.

The ethanol extracts of V. mespilifolia and K. africana gave the highest activity at MIC 2.5 mg/mL against P. aeruginosa; but gave no substantial activity against E. cloacae and S.aureus.

The aqueous extracts of all the tested samples gave the lowest antibacterial activity compared to the organic solvent extracts. However, there was no activity recorded for the aqueous extracts of V. mespilifolia and their combination of all the strain tested. The standard drug

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(Ciprofloxacin) showed greater antibacterial activity against all the tested strains with MIC values ranging from 0.15625 - 2.5 mg/mL.

Extract/Standard MIC (mg/mL) B. A. L. E. P. S. thetaiotomicron odontolyticus Sakei cloacae aeruginosa aureus Acetone combined 5 2.5 5 5 >5 >5

Acetone V. mespilifolia 2.5 2.5 2.5 5 >5 >5

Acetone K. africana >5 >5 >5 >5 >5 >5

Ethanol combined 5 5 5 5 5 >5

Ethanol V.mespilifolia 5 2.5 5 5 2.5 >5

Ethanol K. africana >5 2.5 5 5 2.5 5

Aqueous combined >5 >5 >5 >5 >5 >5

Aqueous V. mespilifolia >5 >5 >5 >5 >5 >5

Aqueous K. africana >5 >5 >5 5 5 5

Ciprofloxacin 0.15625 0.15625 0.15625 0.15625 0.15625 2.5

Anti-fungal activity

Table 6.2 shows the results of the antifungal minimum inhibitory concentration (MIC) of the extracts against selected fungal stains. The acetone and ethanol extracts of the combination of both plants exhibited the highest activity with an MIC of 1.25 mg/mL against M. gypsum while the aqueous extracts of V. mespilifolia, K. africana and their combination showed no antifungal activity M.gypseum. The acetone extracts of V. mespilifolia and K. africana and the ethanol extract of K. africana had the highest activity with an MIC of 0.1325 mg/mL against P.

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chrysogenum; also the aqueous extracts of V. mespilifolia, K. africana and their combination showed no activity against P. chrysogenum. Interestingly, T. tonsurans was resistant to all the extracts used for screening the antifungal activity. The acetone and ethanol extracts of K. africana gave the highest activity against C. albicans with MIC values of 0.3125 mg/mL. The standard drug nystatin showed high antifungal activity with a MIC value ranging from 4 µg/mL to >16 µg/mL.

Table 6. 2. Minimum Inhibitory Concentration (MICs) of the different solvent extracts of V. mespilifolia, K. africana and their combination and nystatin on selected fungal isolates associated with obesity . Extract/standard MIC (mg/mL)

M. P. chrysogenum T. tonsurans C.

gypseum albicans

Acetone combined 1.25 2.5 >5 1.25

Acetone V. mespilifolia 2.5 0.3125 >5 5

Acetone K. africana 5 0.3125 >5 0.3125

Ethanol combined 1.25 1.25 >5 1.25

Ethanol V. mespilifolia 2.5 1.25 >5 5

Ethanol K. africana 5 0.3125 >5 0.3125

Aqueous combined >5 >5 >5 >5

Aqueous V. mespilifolia >5 >5 >5 >5

Aqueous K. africana >5 >5 >5 5

Nystatin 8 4 4 >16

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DISCUSSION There has been considerable interest in exploring the antimicrobial properties of various herbal extracts, as several synthetic antimicrobial drugs used for humans or animals results in the development and propagation of resistant bacteria, and misuse or abuse worsens the situation

(Chang et al. 2015). In addition, the increase in multi-drug resistant micro-oraganism has as lead to the search for new, safe and effective bioactive agents of plant origin (Olajuyigbe and

Afolayan, 2012). Herbal extracts have been proven to be rich in phytochemicals, which are less toxic and more effective in the control of microorganism growth (Ahmad and Beg, 2001).

Herbal remedies have been enjoying increased popularity recently and as such there is need to validate their so called therapeutic potentials using in vitro and in vivo tests on a myriad of micro-organisms (Ashafa, 2013).

In this study, acetone, aqueous and ethanol of V. mespilifolia, K. africana and the combination of both plants inhibited the growth of the selected bacteria which are associated with obesity.

The three plant extracts inhibited the growth of Gram-positive bacteria (A. odontolyticus, L. sakei and S. aureus) and Gram-negative bacteria (B. thetaitotomicron, P. aeruginosa and E. cloacae). Previous studies have shown that plant extracts posses greater efficacy against gram- positive than gram-negative organisms (Lino and Deogracious, 2006; Mudzengi et al. 2017).

This could be attributed to the cell wall of the outer membrane in Gram-negative bacteria whose lipopolysaccharide covering acts as a barrier to many substances including antibiotics

(Klancnik et al. 2010).

The anti-bacterial ability of acetone and ethanol extract of V. mespilifolia was observed to more potent when compared with other extracts tested. In all, the acetone and ethanol extracts of these plants showed better activity against all the bacterial strains tested. These findings are not strange because most antibacterial active components in plant are saturated organic

108

molecules, which are best extracted using non-polar solvents (Mudzengi et al. 2017).

Therefore, from this study it can be deduced that active lipophilic constituents with greater antibacterial activity were best extracted in acetone and ethanol. This implies that these plants possess antibacterial activity against the selected strains and could be crucial in the treatment or management of microbes of the gastrointestinal tract that are associated with obesity complications (Conterno et al. 2011; Lee and Jeon, 2015).

Obesity affects the whole body, and the skin is no exception. Its effect on the skin ranges from dark, velvety patches to red, itchy skin-fold infections, skin tags and even psoriasis, all these are attributed to obesity. Excess body fat leads to extra folds of skin (Lecerf et al. 2003). The combined actions of moisture, warmth and skin rubbing against skin leaves which is highly susceptible to bacterial and fungal infection. Also the development of pimple-type lumps called abscesses beneath the skin in the armpits and groin, results in pain, drainage and deep scarring

(Scheinfeld, 2004). According to Arif et al. (2009), plants possess bioactive secondary metabolites which are active against fungi. The fungitoxic effects of acetone, aqueous and ethanol extracts of V. mespilifolia, K. africana and the combination of both plants in the present study indicates the importance of medicinal plant species as natural sources of antimycotic substances. Antifungal activity of medicinal plants, such as Juglans sp. and Solanum sp. extracts, against some dermatophytes including M. canis and T. mentagrophytes have been reported by other workers (Otang et al. 2012; Parveen et al. 2014). In this study, different plant extracts were tested against four selected opportunistic fungi isolates associated with infections in obese status. The results showed that the plant extracts were active against the growth of only three fungal species. T. tonsurans was resistant to all the extracts used. The acetone and ethanol extracts of V. mespilifolia, K. africana and the combination of both plants were more active against M. gyseum, P. chrysogenum and C. albicans. However, there was no activity

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recorded for the aqueous extracts of V. mespilifolia, K. africana and the combination of both plants. The findings in this study indicate that the different plant extracts of V. mespilifolia, K. africana and the combination of both plants exhibited mild antibacterial activity. Though, strong activity was observed with fungal isolates, promising activities were observed in the acetone and ethanol extracts of the plants tested. This suggests that the plants are important sources of antifungal compounds that could provide renewable sources of useful antifungal drugs against dermatophytic infection associated with obesity.

CONCLUSION The outcome of this study clearly revealed that the acetone and ethanol of extracts of V. mespilifolia, K. africana and the combination of both plants had mild antibacterial and strong antifungal activities which could possibly produce encouraging clinical outcomes. While the aqueous extracts had weak activities against bacterial and anti-fungi. These findings also revealed the multi-pharmacological use of these plants in treatment of dermatophytic infections associated with obesity in South Africa traditional medicine.

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CHAPTER SEVEN

Toxicity evaluation of Vernonia mespilifolia Less (A South Africa medicinal plant) using brine shrimp

This chapter has been published in the Journal of Pharmacology and Toxicology 2017; 12(2): 103–110

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Chapter Seven Toxicity evaluation of Vernonia mespilifolia Less (A South Africa medicinal plant) using brine shrimp

Contents Pages

Abstract...... 118

Materials and Methods ...... 119

Results and Discussion ...... 120

Conclusion ...... 124

References ...... 124

117

OPEN ACCESS Journal of Pharmacology and Toxicology

ISSN 1816-496X DOI: 10.3923/jpt.2017.103.110

Research Article Toxicity Evaluation of Vernonia mespilifolia Less (A South Africa Medicinal Plant) Using Brine Shrimp

Jeremiah Oshiomame Unuofin, Gloria Aderonke Otunola and Anthony Jide Afolayan

Medicinal Plants and Economic Development (MPED) Research Centre, Department of Botany, University of Fort Hare, 5700 Alice, South Africa

Abstract Background and Objective: Vernonia mespilifolia is widely used in folk medicine in the Eastern Cape province, South Africa. This study evaluated the toxicity of acetone, aqueous and ethanol extracts of Vernonia mespilifolia using brine shrimp model. Materials and Methods: Different concentrations (0.0625-1 mg mLG1) of the extracts were used to incubate the cysts and nauplii (hatched cysts) of brine shrimp (Artemia salina) to evaluate their effects on the hatching success of the cyst and lethality of the nauplii respectively. The percentage hatching success of cyst and Lethal Concentration (LC50) to kill 50% of the nauplii were recorded. Results: The hatching success was in order: Aqueous extract (48.6%)>acetone extract (38.2%)>ethanol extract (26.8%). The hatching of nauplii was in a concentration dependent fashion, with hatching success decreasing with increase in concentration of extracts. Lethality 1 of extract was based on Meyer’s index of toxicity. Conclusion: All the three extracts showed high levels of toxicity with LC50 <1 mg mLG which signify toxicity in a brine shrimp model. In this respect, V. mespilifolia possesses cytotoxic behavior suggesting the presence of potential bioactive chemical component in the plant extract. Further in vivo and cell lines cytotoxicity test is recommended to substantiate these findings.

Key words: Vernonia mespilifolia, toxicity, brine shrimp, hatchability, lethality, nauplii, cyst, extracts

Received: November 08, 2016 Accepted: February 13, 2017 Published: March 15, 2017

Citation: Jeremiah Oshiomame Unuofin, Gloria Aderonke Otunola and Anthony Jide Afolayan, 2017. Toxicity evaluation of Vernonia mespilifolia Less (A South Africa medicinal plant) using brine shrimp. J. Pharmacol. Toxicol., 12: 103-110.

Competing Interest: The authors have declared that no competing interest exists.

Data Availability: All relevant data are within the paper and its supporting information files. 118 J. Pharmacol. Toxicol., 12 (2): 103-110, 2017

INTRODUCTION Although V. mespilifolia is used for ethnomedicinal purposes, there is limited knowledge about it toxicity level. The pivotal role medicinal plants and traditional health This study aim to investigate the potential toxicity of the crude systems play in solving the health care problems of the world extracts of Vernonia mespilifolia using brine shrimp model. is gaining increasing attention. As a result of this rebirth of interest, study on medicinal plants is rising impressively at the MATERIALS AND METHODS international level, particularly in developing countries where traditional medical practice is imbibed as an essential part of The whole plant parts used for this study was collected in their culture1. Bioactive compounds present in medicinal June, 2015 from its natural habitat in the wild at Zihlahleni plants are responsible for their efficacy2. These compounds are village Maipase, Nkonkobe Municipality of the Eastern Cape, mainly secondary metabolites and they include alkaloids, South Africa; which lies at latitude 32E51N 41.846O S and essential oils, tannins and resins to mention a few, which longitude 7E 10N 59.318O E. The plant was authenticated by function either in their original form or in semi-synthetic Mr. Tony Dold of Selmar Schonland Herbarium, Rhodes 3 forms . In spite of these bioactive compounds exhibiting University, South Africa and a voucher specimen (Unuofin therapeutic potential, there is insufficient knowledge Med, 2015/1) was prepared and deposited at the Giffen about their toxicogenic effects when consumed in large Herbarium, University of Fort Hare. amounts4. Many research studies at present focus on both pharmacology and toxicity of medicinal plants used by Preparation of extracts: The whole plant was rinsed with humans to promote safety with the use of plant products for deionised water and gently blotted dry with paper towel and the treatment of various ailments5. subsequently oven-dried (LABOTEC, South Africa) at 55EC for To this end, it is of great importance to verify the 72 h until constant weight was achieved. The dried sample pharmacological qualities of herbal-derived remedies and also was then ground into powder (Polymix® PX-MFC 90D their level of toxicity contrary to the putative view of the Switzerland) and stored at 4EC till needed for analyses. The innocuity/innocuousness of natural products6. ground sample (200 g) was weighed into 3 separate conical Various assays are being employed for the study of flasks containing (2 L) acetone, ethanol and water respectively potential toxicity of herbal extracts based on different and shaken in an orbital shaker (Orbital incubator shaker, biological models, such as in vivo assays on laboratory Gallenkamp) for 48 h. The crude extracts were filtered under animals. Brine Shrimp Lethality Assay (BSLA) has gained pressure using a Buchner funnel and Whatman No. 1 filter recognition as an alternative bioassay technique to screen the paper. The acetone and ethanol extracts were further toxicity of algae7, dental materials8, heavy metals9 and metal concentrated to dryness to remove the solvents under ions10, toxicity of nanoparticles11, as well as screening of reduced pressure using a rotary evaporator (Strike 202 marine natural products12 and the toxicity plant extracts13-16. Steroglass, Italy), while the aqueous filtrate obtained was It also indicated cytotoxicity of a myriad of pharmacological concentrated using a freeze dryer (Vir Tis benchtop K, Vir Tis activities such as antimicrobial, antiviral, pesticidal and anti-tumor of compounds13,14. Co., Gardiner, NY). Vernonia mespilifolia Less., popularly known as Uhlunguhlungu (Xhosa) among the indigenous people of the Artemia salina hatching assay: The method described by 21 Nkonkobe Municipality of the Eastern Cape, South Africa is Otang et al. was employed with little modifications. Five petri one of the five Southern African species of the Vernonia dishes containing 30 mL of the extracts were prepared in genus that is endemic or near-endemic to this filtered sea water by dissolving them in minute amount (1 mL) subcontinent17. It is a climbing shrub that is 0.6-9.0 m tall, of the parent solvents to yield a two-fold dilution of with pinnately-veined leaves, epaleate receptacle with concentrations (1, 0.5, 0.25, 0.125 and 0.0625 mg mLG1). A obtuse involucral bracts and white to violet florets17. positive control was also prepared by dissolving potassium Vernonia mespilifolia is commonly found in the Eastern Cape, dichromate in seawater in the same concentrations as the Kwazulu-Natal, Limpopo, Mpumalanga and Western Cape plant extracts. Sea water alone was used as the negative provinces of South Africa18. It is used in the Eastern Cape of control. The setup was allowed to stand for 30 min to allow South Africa for ethnomedicinal management of weight loss the solvents to evaporate. and hypertension19 and also for the treatment of heart water Ten A. salina cysts were stocked in each of the petri disease in goats20. dishes containing 30 mL of the prepared two-fold

119 J. Pharmacol. Toxicol., 12 (2): 103-110, 2017 concentrations (1-0.0625 mg mLG1) of the plant fractions and was done on MINITAB version 17 for windows. One-way positive control. The petri dishes were partly covered, analysis of variance (ANOVA) followed by Fischer’s least incubated at 30EC and under constant illumination for 72 h. significant difference (for means separation) was used to test The number of free nauplii in each petri dish was counted the effect of concentration and time of exposure of the plant after every 24 h till the end of 72 h. The percentage of extracts on the hatchability success of the cysts and mortality hatchability was calculated by comparing the number of of larvae respectively. hatched nauplii with the total number of cysts stocked. RESULTS AND DISCUSSION Artemia salina lethality assay: Artemia salina cysts were hatched in sea water and 10 nauplii were pipetted into each Brine shrimp hatchability: Brine shrimp hatchability test was petri dish containing the two-fold concentrations of the used to determine the biological activity of Vernonia extracts and control as in the hatchability assay described mespilifolia. The hatching success of A. salina incubated with above. The petri dishes were then examined and the number different plant extracts and control is as shown in Fig. 1. The of living nauplii (that exhibited movement during several sea water exhibited a significantly higher (p<0.05) hatching seconds of observation) was counted after every 24 h and success (71%) than the solvent extracts and the positive the set up was allowed to stand for 72 h under constant control (potassium dichromate) (5.4%). The hatching success illumination. The percentage of mortality (M%) was of the cysts in the acetone, aqueous and ethanol extracts calculated as following in Eq. 1: were 38.2, 48.6 and 26.8%, respectively and were significantly different (p<0.05) from each other. The hatching success of Total nauplii Live nauplii A. salina cysts incubated with aqueous extracts had the Mortality (%) = ×100 (1) Total nauplii highest hatching success and presumably least toxic of the solvent extracts used (Fig. 1). This could explain why most Data analysis: The percentage hatchability success and traditional herbal medicines are prepared using water as a mortality data obtained from the 5 different concentrations of solvent because it is not or less toxic. This could also suggest each fraction and control experiments were used to construct why the cyst showed more resistant to hatching in the the dose-response curves. These were used to determine their acetonic and ethanol extracts than in the aqueous extracts. corresponding LC50 values. The LC50 was taken as the This resistant could be attributed to the permeability barrier concentration required for producing 50% mortality of the provided by the cysts22,23. nauplii. The LC50 values were determined from the best-fit line The A. salina embryos are highly defenseless to toxins obtained by regression analysis of the percentage hatchability at early developmental stages24-26. In the brine shrimp and lethality versus the concentration. The statistical analysis hatchability assay, the hatching success significantly

80 e 70 )

% 60

50 a b 40

30 c

20 Hatchability success ( Hatchability

10 d 0

ct te er ract ra a t t m wat extract x ex ro l ea e e o ich S an d eton h m c t u Aqueous A E si tas o P

Fig. 1: Percentage hatching success of A. salina cysts incubated in different solvent extracts and controls Values are means of 5 concentrations for each plant extract/control ±SD of three replicates. Bars with different letters are significantly different at p<0.05

120 J. Pharmacol. Toxicol., 12 (2): 103-110, 2017

90 Aqueous extract Ethanol extract Acetone extract Potassium dichromate 80 a

70 a a b 60 a 50 b c b 40 b

30 a a

Hatchability success (%) a b 20 b 10 c c c c 0 0 0.0625 0.125 0.25 0.5 1 Concentration (mg mLG1 )

Fig. 2: Effect of varying concentrations on hatching success (%) of A. salina cysts Values are Means±SD of three replicates of the concentrations for each plant extract/control. Set of bars with different letters are significantly different at p>0.05

Aqueous extract Sea water 90 Acetone extract Potassium dichromate Ethanol extract c c c c 80 70

) b 60 a a % a a 50 a a b 40 b a b b b a Hatchability ( 30 b a.c 20 d c 10 d dd 0 24 36 48 60 72 Time (h) Fig. 3: Effect of time (h) on the hatching success of A. salina cysts Values are Means±SD of three replicates for each plant extract/control. Set of bars with different letters are significantly different at p<0.05 decreased with increasing concentrations of the crude no significant difference in their hatching success at extracts in a dose dependent manner. The hatching success of 0.25 mg mLG1. At concentrations of 0.25-1 mg mLG1 for the cyst was also evaluated at different concentrations and the potassium dichromate, no hatching was observed. Also, result is depicted in Fig. 2. The aqueous extract exhibited its cysts incubated in 1 mg mLG1 of acetone and ethanol extracts maximum hatching success at 0.5 mg mLG1 (76%). The did not hatch. The inability of cysts to hatch in V. mespilifolia acetone and ethanol extracts had similar hatching pattern, crude extracts could be attributed to the toxic compounds with the highest hatching success observed at 0.125 mg mLG1 present in the extracts that have ovicidal property. Also, (Fig. 2). The overall best hatching success potential was reports have shown that secondary metabolites in plants observed with cysts incubated at 0.5 mg mLG1 in the aqueous could have effect on the embryonic development27. extract which was significantly (p>0.05) greater when The results of the effect of exposure time on hatchability compared with other solvents used. Potassium dichromate showed that the sensitivity of A. salina cysts to the plant showed the lowest hatching success. At 0.0625 mg mLG1, extracts was strongly dependent upon exposure period there was no significant difference in the hatching success of (Fig. 3). A similar trend was observed with the aqueous and aqueous extract and potassium dichromate. There was also no acetone extracts as there was significant differences in significant difference in the hatching success of cysts hatching success from 24-48 h (p>0.05). The lowest hatching incubated at 0.125 mg mLG1 in both the aqueous and success was observed after 24 h treatment in all the extracts. acetone extracts. The acetone and ethanol extracts had This result is in line with reports from Vasconcelos et al.26 and

121 J. Pharmacol. Toxicol., 12 (2): 103-110, 2017

120

d 100

80 a a 60 b

Mortality (%) Mortality 40

c 0

ct ct er te ract ra ra t t t wat ma x x ex o s e e hr ne Sea ic ou to anol e e h qu c m d A A Et siu tas Po

Fig. 4: Percentage mortality of A. salina nauplii incubated in different plant extracts and controls Values are means of 5 concentrations for each plant extract/control ±SD of three replicates. Bars with different letters are significantly different at p<0.05

120 Aqueous extract Ethanol extract Acetone extract Potassium dichromate b c c b b 100 d a a a 80 a a a 60 a b b a Mortality (%) 40 a b a 20 c

0 0.0625 0.125 0.25 0.5 1 Concentration (mg mLG1 )

Fig. 5: Percentage mortality of A. salina cysts incubated at different concentrations of the plant extracts and control Values are means of three replicates ±SD (at different hours). Set of bars with different letters are significantly different at p<0.05

Subhadra et al.27, which stated that A. salina is extremely determines lethal concentration of active compounds such as susceptible to toxin during its early stage of development. heavy metals, pesticides and medicines in brine medium28-30 The cysts in acetone and ethanol extracts experienced and has been employed to determine toxicity of various maximum hatching at 48 h with 45 and 32% hatching success active compounds because it is reliable, rapid and very respectively, after which death set in for the already hatched convenient to carry out13,31,21. The percentage mortality of nauplii. The aqueous extract had the highest hatching success A. salina larvae (nauplii) incubated in different solvent at 60 h. Also, in Fig. 3 there was a 1.3 and 1.1 fold decrease extracts of Vernonia mespilifolia and controls are shown in respectively in hatched cyst from 48-72 h in both acetone and Fig. 4. There was high mortality of nauplii incubated in both ethanol extracts, while aqueous extract decreased significantly the aqueous and acetone extracts, although there was no (p<0.05) by 1.1 fold after 60 h. Sea water exhibited optimum significant difference in the percentage mortality, whereas the hatching at 36 h and remained the same throughout the nauplii incubated with potassium dichromate had a experiment. Hatching of cysts incubated in potassium significantly greater mortality when compared to all the dichromate decreased significantly by 3.5-folds after 36 h extracts and sea water (p<0.05). The ethanolic extract and sea (p<0.05), followed by no hatching of cysts after 48 h. water showed a mortality of 49.60 and 0%, respectively. The effect of different concentrations of the plant extracts Brine shrimp lethality: Brine shrimp cytotoxicity test is on the mortality of larvae is shown in Fig. 5. The degree of considered as a preliminary assessment of toxicity. This assay mortality of nauplii was in a concentration-dependent

122 J. Pharmacol. Toxicol., 12 (2): 103-110, 2017

120 Aqueous extract Sea water Acetone extract Potassium dichromate Ethanol extract c d d e d 100 a a a b 80 b

a.b a a c 60 a b Mortality (%) Mortality 40 b a a a

c c 0 b d c 24 36 48 60 72 Time (h)

Fig. 6: Percentage mortality of A. salina cysts incubated at different time durations in the plant extracts and controls Values are Means±SD of 3 replicates (of all the concentrations) for each plant extract/control ±SD. Set of bars with different letters are significantly different at p<0.05

Table 1: Lethal dose concentration (LC50) of acetone, ethanol and aqueous extracts of Vernonia mespilifolia against brine shrimp 1 2 Sample Regression equation LC50 (µg mLG ) Toxicity status R (%) Aqueous extract Y = 19.476ln(x)+89.4 132 Toxic 97.4 Acetone extract Y = 33.161x+47.75 67.8 Toxic 85.8 Ethanol extract Y = 88.667x+16.042 383 Toxic 96.3 Potassium dichromate Y = 1.4427247ln(x)+101 <0.100 Toxic 50 1 LC50 is the concentration (µg mLG ) of the plant extracts and positive control (Potassium dichromate) sufficient to obtain 50% of inhibition of nauplii mortality of A. salina, respectively. R2 is the coefficient of determination of the regression equation fashion. The highest mortality was observed in all the extracts observed that between 24-72 h of exposure of the nauplii at 1 mg mLG1 compared to potassium dichromate which to aqueous, acetone and ethanol extracts, there were 2.94, showed maximum mortality (100%) at 0.125 mg mLG1. 3.41 and 2.5-folds increase in the mortality of nauplii, There was no significant difference (p<0.05) in percentage respectively. The nauplii incubated in sea water did not die mortality of the nauplii between the aqueous extract throughout the duration of the experiment. Generally, the and acetone extract at concentrations of 0.125, 0.25 mortality of nauplii was significantly similar when and 1 mg mLG1. At 0.125 and 0.5 mg mLG1, aqueous and incubated with the ethanol, acetone and the aqueous ethanol extracts also exhibited no significance difference in extracts at 24 h and was significantly higher than sea percentage mortality (p<0.05). At 1 mg mLG1, there was no water (p<0.05) (Fig. 6). The mortality of nauplii incubated in significance difference in percentage mortality between these plant extracts increased exponentially with time aqueous and acetone extracts and also between the with the highest mortality observed at 72 h with all the ethanolic extract and potassium dichromate (p<0.05) in extracts. The nauplii attain the second and third instars of their Fig. 5. The results revealed that the effect of varying life cycle within 48 h, hence their greatest sensitivity to toxins concentrations of all the plant extracts on the mortality of at this time34,35. However, the findings of this study indicated larvae was in a concentration dependent fashion, therefore it that the maximum sensitivity was reached after 72 h of can be postulated that though these are toxicological data, exposure. this plant possesses pharmacological activity based on the According to Meyer et al.17 and Bastos et al.36 the dosage administered21,32. brine shrimp lethality were interpreted in accordance

All extracts were screened at 5 different concentrations to the criterion by Meyer toxicity index that LC50 values 1 1 1 viz., 62.5, 125, 250, 500 and 1000 µg mLG and observed for >1000 µg mLG (1 mg mLG ) are considered non-toxic, LC50 their toxic effect on A. salina rom 24-72 h. Potassium values >500 µg mLG1 (0.5 mg mLG1) but not >1000 µg mLG1 dichromate was used as a standard33. are considered to have weak toxicity, while those having 1 The percentage mortality due to exposure time is as LC50 values <500 µg mLG are considered toxic. The LC50 shown in Fig. 6. The result revealed that the percentage values were calculated as 132, 6.78 and 383 µg mLG1 for mortality was time dependent; the longer the exposure of aqueous extract, acetone extract and the ethanol extracts, nauplii to the plant extracts, the greater the mortality. It was respectively (Table 1).

123 J. Pharmacol. Toxicol., 12 (2): 103-110, 2017

The BSLA result of all the crude extracts of V. mespilifolia REFERENCES 1 showed that the extracts were toxic with LC50 <1 mg mLG (Table 1); hence these extracts may not be considered safe for 1. Robinson, H. and V.A. Funk, 2014. Gymnanthemum consumption as herbal medicine. These toxic results from this koekemoerae (Compositae, Vernonieae), a new species from study could be employed as promising alternative in the South Africa. PhytoKeys, 30: 59-65. treatment and management of tumors as brine shrimp 2. Raimondo, D., L. von Staden, W. Foden, J.E. Victor and lethality test now serves as an indicator for the preliminary N.A. Helme et al., 2009. Red List of South African Plants. screening of bioactivity including for anticancer37 properties. Strelitzia 25. South African National Biodiversity Institute, The combination of larval mortality and increased exposure Pretoria. 3. Afolayan, A.J. and B.O. Mbaebie, 2010. Ethnobotanical period further improved the bioassay sensitivity, providing study of medicinal plants used as anti-obesity remedies values which are typically lower than embryotoxicity assays. in Nkonkobe Municipality of South Africa. Pharmacogn. The use of Artemia salina 72 h larval mortality assay may be J., 2: 368-373. used as an alternative bioassay to screen for toxic effects of 4. Dold, A.P. and M.L. Cocks, 2001. Traditional veterinary novel bioactive compounds, particularly in situations where medicine in the Alice district of the Eastern Cape Province, the availability of mature invertebrates such as sea urchins is South Africa. S. Afr. J. Sci., 97: 375-379. problematic. 5. Farnsworth, N.R. and D.D. Soejarto, 1991. Global Importance of Medicinal Plants. In: Conservation of Medicinal Plants, CONCLUSION Akerele, O., V. Heywood and H. Synge (Eds.). Cambridge University Press, Cambridge, New York, pp: 25-51. The brine shrimp lethality bioassay now serves as a useful 6. Salim, A.A., Y.W. Chin and A.D. Kinghorn, 2008. Drug tool for the preliminary assessment of toxicity from plant Discovery from Plants. In: Bioactive Molecules and Medicinal extracts. It can be deduced that the acetone, aqueous and Plants, Ramawat, K.G. and J.M. Merillon (Eds.)., Chapter 1. ethanol extracts of Vernonia mespilifolia contains certain Springer-Verlag, Berlin, Heidelberg, pp: 1-24. 7. Hassan, B.A.R., 2012. Medicinal plants (Importance and uses). useful bioactive compounds which though toxic can be Pharm. Anal. Acta, Vol. 3. 10.4172/2153-2435.1000e139. harnessed and purified into useful therapeutic drugs. 8. Cupp, M.J., 1999. Herbal remedies: Adverse effects and drug Although the brine shrimp lethality cytotoxicity bioassay is interactions. Am. Family Phys., 59: 1239-1245. rather insufficient as to the elucidation of the mechanism of 9. Parra, A.L., R.S. Yhebra, I.G. Sardinas and L.I. Buela, 2001. action. Comparative study of the assay of Artemia salina L. and the estimate of the medium lethal dose (LD50 value) in mice, SIGNIFICANT STATEMENT to determine oral acute toxicity of plant extracts. Phytomedicine, 8: 395-400. This study sought to screen the potential toxicity of 10. Firenzuoli, F. and L. Gori, 2007. Herbal medicine today: Clinical Vernonia mespilifolia Less., a medicinal plant widely used for and research issues. Evid. Based Complement. Alternat. Med., the management of weight loss and hypertension in 4(Suppl. 1): 37-40. South Africa using the brine shrimp toxicity assay. The findings 11. Mayorga, P., K.R. Perez, S.M. Cruz and A. Caceres, 2010. revealed that all the solvent extracts screened were toxic. Comparison of bioassays using the anostracan crustaceans Therefore, these results indicate that V. mespilifolia may not Artemia salina and Thamnocephalus platyurus for plant extract toxicity screening. Rev. Bras. Farmacogn., 20: 897-903. be safe for human consumption. However, further studies are 12. Pelka, M., C. Danzl, W. Distler and A. Petschelt, 2000. A new on-going to establish the toxicity and its alternative use in screening test for toxicity testing of dental materials. cancer cells. J. Dentistry, 28: 341-345. 13. Martinez, M., J.D. Ramo, A. Torreblanca and J. Diaz-Mayans, ACKNOWLEDGMENT 1999. Effect of cadmium exposure on zinc levels in the brine shrimp Artemia parthenogenetica. Aquaculture, 172: 315-325. Authors wish to acknowledge the financial support of 14. Kokkali, V., I. Katramados and J.D. Newman, 2011. Monitoring Govan Mbeki Research and Development Centre, University of the effect of metal ions on the mobility of Artemia salina Fort Hare, Eastern Cape, South Africa. Nauplii. Biosensors, 1: 36-45.

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15. Maurer-Jones, M.A., S.A. Love, S. Meierhofer, B.J. Marquis, 27. Subhadra, S., V.R. Kanacharalapalli, V.K. Ravindran, S.K. Parre, Z. Liu and C.L. Haynes, 2013. Toxicity of nanoparticles to brine S. Chintala and R. Thatipally, 2011. Comparative toxicity shrimp: An introduction to nanotoxicity and interdisciplinary assessment of three Tephrosia species on Artemia salina science. J. Chem. Educ., 90: 475-478. and animal cell lines. J. Nat. Pharm., 2: 143-148. 16. Carballo, J.L., Z.L. Hernandez-Inda, P. Perez and 28. Solis, P.N., C.W. Wright, M.M. Anderson, M.P. Gupta and M.D. Garcia-Gravalos, 2002. A comparison between two brine J.D. Phillipson, 1993. A microwell cytotoxicity assay using shrimp assays to detect in vitro cytotoxicity in marine natural Artemia salina (brine shrimp). Planta Med., 59: 250-252. products. BMC Biotechnol., Vol. 2. 10.1186/1472-6750-2-17. 29. Jaki, B., J. Orjala, H.R. Burgi and O. Sticher, 1999. 17. Meyer, B.N., N.R. Ferrigni, J.E. Putnam, L.B. Jacobsen, Biological screening of cyanobacteria for antimicrobial and D.E. Nichols and J.L. McLaughlin, 1982. Brine shrimp: A convenient general bioassay for active plant constituents. molluscicidal activity, brine shrimp lethality and cytotoxicity. Planta Med., 45: 31-34. Pharm. Biol., 37: 138-143. 18. McLaughlin, J.L., L.L. Rogers and J.E. Anderson, 1998. The 30. Piccardi, R., A. Frosini, M.R. Tredic and M.C. Margheri, 2000. use of biological assays to evaluate botanicals. Drug Inform. Bioactivity in free-living and symbiotic cyanobacteria of the J., 32: 513-524. genus Nostoc. J. Applied Phycol., 12: 543-547. 19. Moshi, M.J., E. Innocent, J.J. Magadula, D.F. Otieno, 31. Lahti, K., J. Ahtiainen, J. Rapala, K. Sivonen and S.I. Niemela, A. Weisheit, P.K. Mbabazi and R.S.O. Nondo, 2010. Brine 1995. Assessment of rapid bioassays for detecting shrimp toxicity of some plants used as traditional medicines cyanobacterial toxicity. Lett. Applied Microbiol., 21: 109-114. in Kagera region, North Western Tanzania. Tanz. J. Health Res., 32. Kibiti, C.M. and A.J. Afolayan, 2016. Antifungal activity and 12: 63-67. brine shrimp toxicity assessment of Bulbine abyssinica 20. Sharma, N., P.C. Gupta, A. Singh and C.V. Rao, 2013. Brine used in the folk medicine in the Eastern Cape Province, shrimp bioassay of Pentapetes phoenicea Linn. and Ipomoea South Africa. Bangladesh J. Pharmacol., 11: 469-477. carnea Jacq. leaves. Der Pharmacia Lettre, 5: 162-167. 33. Padmaja, R., P.C. Arun, D. Prashanth, M. Deepak, A. Amita and 21. Otang, M.W., S.D. Grierson and N.R. Ndip, 2013. Assessment of M. Anjana, 2002. Brine shrimp lethality bioassay of selected potential toxicity of three South African medicinal plants using the brine shrimp (Artemia salina) assay. Afr. J. Pharm. Indian medicinal plants. Fitoterapia, 73: 508-510. Pharmacol., 7: 1272-1279. 34. Lewis, G.E., 1995. Testing the toxicity of extracts of Southern 22. Adwan, K. and N. Abu-Hasan, 1998. Gentamicin resistance in African plants using brine shrimp (Artemia salina). S. Afr. J. clinical strains of Enterobacteriaceae associated with reduced Sci., 91: 382-384. gentamicin uptake. Folia Microbiol., 43: 438-440. 35. Sreejamole, K.L. and C.K. Radhakrishnan, 2013. Antioxidant 23. Abu-Shanab, B., G. Adwan, D. Abu-Safiya, N. Jarrar and and cytotoxic activities of ethyl acetate extract of the K. Adwan, 2004. Antibacterial activities of some plant Indian green mussel Perna viridis. Asian J. Pharm. Clin. extracts utilized in popular medicine in Palestine. Turk. J. Biol., Res., 6: 197-201. 28: 99-102. 36. Bastos, M.L.A., M.R.F. Lima, L.M. Conserva, V.S. Andrade, 24. Sleet, R.B. and K. Brendel, 1985. Homogeneous populations E.M.M. Rocha and R.P.L. Lemos, 2009. Studies on the of Artemia nauplii and their potential use for in vitro testing antimicrobial activity and brine shrimp toxicity of in developmental toxicology. Teratog. Carcinog. Mutagen., Zeyheria tuberculosa (Vell.) Bur. (Bignoniaceae) extracts 5: 41-54. and their main constituents. Ann. Clin. Microbiol. Antimicrob., 25. Lewan, L., M. Andersson and P. Morales-Gomez, 1992. Use of Artemia salina in toxicity testing. Altern. Lab. Anim., Vol. 8. 10.1186/1476-0711-8-16. 20: 297-301. 37. Artanti, N., T. Firmansyah and A. Darmawan, 2012. 26. Vasconcelos, V., J. Azevedo, M. Silva and V. Ramos, 2010. Bioactivities evaluation of Indonesian mistletoes Effects of marine toxins on the reproduction and early stages (Dendrophthoe pentandra (L.) Miq.) leaves extracts. development of aquatic organisms. Mar. Drugs, 8: 59-79. J. Applied Pharm. Sci., 2: 24-27.

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CHAPTER EIGHT

Toxicity assessment of Kedrostis africana: A medicinal plant used in the management of obesity in South Africa using brine shrimp assay

This chapter has been accepted in this format in International Journal of Pharmaceutical Science and Research

126

Chapter Eight Toxicity assessment of Kedrostis africana cogn: A medicinal plant used in the management of obesity in south africa using brine shrimp assay.

Contents Pages

Abstract ...... 128

Introduction ...... 129

Materials and methods ...... 130

Result ...... 132

Discussion ...... 139

Conclusion ...... 141

References ...... 142

127

ABSTRACT Baboon's Cucumber (Kedrostis africana) is a monoecious caudiciform plant that belongs to

Cucurbitaceae family. Baboon's Cucumber is used traditionally for the management of syphilis and obesity in South Africa. In the present study, we examined the hatchability and lethality of

Baboon's Cucumber bulb extracts against brine shrimps. The tested samples were aqueous extract, acetone extract, and ethanol extract. Cytotoxicity was screened using Brine Shrimp

Lethality Test (BSLT). The hatching success was in the order: ethanol extracts (49.2%)

>aqueous extract (45.4%) >acetone extract (45.2%). All the extracts hatching success were significantly higher than the positive control (potassium dichromate) (p <0.05). Based on

Clarkson’s toxicity index, LC50> 1 mg/mL were considered non-toxic for acetone extract while the aqueous and ethanolic extracts were considered to be moderately toxic (LC50 100-500

μg/mL) with LC50 of 0.298 and 0.489 mg/mL respectively.

In conclusion, since the aqueous and ethanolic bulb extracts of K. africana exhibits potent cytotoxic property comparable to that of standard drug. Therefore, this might be utilized for the development of novel anticancer drug leads and the nontoxic acetonic extracts could further be exploited for the development of plant-based pharmaceuticals.

Keyword: Kedrostis africana, toxicity, hatchability, lethality, Artemia salina, extracts.

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INTRODUCTION The healing potentials in plants are an antique concept. The World Health Organization

(WHO) has long recognized and drawn the attention of many countries to the ever increasing interest of the public in the use of medicinal plants and their products in the management or treatment of various ailments. This discovery has brought about the search for novel drugs which are plant-derived and possess the potency to combat the menace of drug resistance pathogenic microorganism, antitumor, anti-obesity and anticancer agents 1, 2. Obesity is becoming one of the most prevalent health concerns among all populations and age groups worldwide, resulting in a significant increase in mortality and morbidity related to coronary heart diseases, diabetes type 2, metabolic syndrome, stroke and cancers 3-5. Recently, there has been difficulty in the treatment of obesity because some of the drugs in the market have side effects, including increased blood pressure, dry mouth, constipation, headache, and insomnia

6, 7. Therefore, it has become imperative to explore natural products for treating obesity as this may be an excellent alternative strategy for developing future effective, safe anti-obesity drugs which would also be affordable 8,9.

Kedrostis africana (Linnaeus) Cogn. is among the plants used by traditional healer for the management of obesity in the Eastern Cape of South Africa by a survey conducted by Afolayan and Mbaebie 10.

Kedrostis africana is a monoecious caudiciform plant, commonly known as "Baboon's

Cucumber" with lots and lots of herbaceous climbing or creeping vines growing rapidly from the swollen base and looking like English ivy with a tuber. The shoots emerge from a massive underground tuberous rootstock (or caudex). This tuber is a water-storage organ so it is very resistant to drought 11. Kedrostis africana is found in Namibia and South Africa (Eastern Cape,

Free State, Gauteng, KwaZulu-Natal, Limpopo, Mpumalanga, Northern Cape, North West, and

Western Cape). According to vanWyk 12, Kedrostis africana tuber is used as an emetic, 129

purgative, diuretic; dropsy, syphilis. Also, a decoction from the crushed fresh bulb is used for the management of obesity 10, 13. Brine shrimp also known as sea monkey is now being used for toxicity assay because this assay involves the killing, thus there have been a reasonable controversy over the use of animal for such purposes and some people have ethical or religious objection to the killing of even lower organism 14. Brine shrimp (Artemia salina) assay is now being employed because it gives a valid method of evaluating the cytotoxic of plant extracts 15-

17. Some publications have suggested a good correlation between the toxic activity in the brine shrimp assay and the cytotoxicity against tumor cell lines 18 and hepatotoxic activity 19. Brine shrimp tests are normally conducted to draw inference on the safety of the plant extracts and further to depict trends of their biological activities. Although the ethno-medicinal purpose has been explored, there is a need to investigate its toxicity level. The aim of the present work is to

Kedrostis africana for its cytotoxic effect on Artemia salina and correlate toxicity results with its known ethnopharmacological activity.

MATERIALS AND METHODS The bulb of K. africana was used for this study were collected August 2015 near baddford farm in Fort Beaufort which is in Raymond Mhlaba Municipality, Eastern Cape, South Africa. This area lies at Latitude 32°43'28.66" and Longitude 26°34'5.88". The plant was authenticated by

Mr. Tony Dold of Selmar Schonland Herbarium, Rhodes University, South Africa, and a voucher specimen (Unuofin Med, 2015/2) was prepared and deposited in the Giffen

Herbarium, University of Fort Hare. The bulb was rinsed with deionised water and gently blotted with paper towel to remove the water, chopped into smaller bits and subsequently oven- dried (LABOTEC, South Africa) at 55°C for 72 hours until constant weight was achieved, then ground into powder (Polymix® PX-MFC 90D Switzerland). The ground sample was put into separate conical flasks containing acetone, ethanol and water and shaken in an orbital shaker

(Orbital Incubator Shaker, Gallenkamp) for 48 hours. The crude extracts were filtered using a 130

Buchner funnel and Whatman No. 1 filter paper. The acetone and ethanol extracts were further concentrated to dryness to remove the solvents under reduced pressure using a rotary evaporator (Strike 202 Steroglass, Italy) while the aqueous filtrate obtained was concentrated using a freeze dryer (Vir Tis benchtop K, Vir Tis Co., Gardiner, NY).

Preparation of the assay

The method described by Otang et al., 20 was employed with little modifications. Five petri dishes containing 30 mL of the extracts were prepared in filtered sea water by first dissolving them in minute amount of the parent solvents to yield a two-fold dilution series of concentrations (1, 0.5, 0.25, 0.125 and 0.0625 mg/mL). A positive control was also prepared by dissolving potassium dichromate in seawater in the same concentrations as the plant extracts. Sea water only served as the negative control. The setup was allowed to stand for 30 minutes to allow the solvents to evaporate.

A. salina hatching assay

This assay was evaluated as described by Otang et al 20. A density of ten (10) A. salina cysts was stocked in each of the petri dishes containing 30 mL of the prepared two-fold concentrations (1 to 0.0625 mg/mL) of the plant fractions and positive control. The petri dishes were partly covered, incubated at 30°C and under constant illumination for 72 hours. The number of free nauplii in each petri dish was counted after every 24 hours till end of 72 hours.

The percentage of hatchability was assessed by comparing the number of hatched nauplii with the total number of cysts stocked.

A. salina lethality assay

131

A. salina cysts were hatched in sea water and 10 nauplii were pipetted into each petri dish containing the two-fold concentrations of the extracts and control as in the hatchability above.

The petri dishes were then examined and the number of living nauplii (that exhibited movement during several seconds of observation) was counted after every 24 hours and the set up was allowed to stand for 72 hours under constant illumination. The percentage of mortality (M %) was calculated as: Mortality (%) = (Total nauplii – Alive nauplii) × 100 / Total nauplii.

Data analysis

The percentage hatchability success and mortality data obtained from the 5 different concentrations of each fraction and control experiments were used to construct the dose- response curves. These were used to determine their corresponding LC50 values. The LC50 was taken as the concentration required for producing 50% mortality of the nauplii. LC50 values were determined from the best-fit line obtained by regression analysis of the percentage hatchability and lethality versus the concentration. The statistical analysis was done on

MINITAB version 17 for windows. One-way analysis of variance (ANOVA) followed by

Fischer’s Least Significant Different (for means separation) was used to test the effect of concentration and time of exposure of the plant extracts on the hatchability success of the cysts and mortality of and larvae respectively.

RESULT

Brine shrimp hatchability assay

The hatching success of A. salina incubated with different plant extracts of K.africana and control is as shown in Figure 8.1 with the sea water having a significantly higher hatching success (71%) than the solvent extracts including the positive control (potassium dichromate)

132

(5.4%) (p< 0.05). The hatching success of the cysts in the acetone (45.2%), aqueous (45.4%) and ethanol (49.2%) extracts showed no significant difference from each other (p < 0.05).

80 c 70

50 a a a

Hatchability (%) Hatchability 20

10 b

0 Aqueous Acetone Ethanol Potassium Sea water extract extract extract dichromate

The effect of different solvent concentrations on the hatching success of the cyst was also evaluated and the result is depicted in Figures 8.2. Figure 8.2 shows the activities of the different plant extracts/positive control at varying concentrations to the hatching success of the cysts. The hatching success of A. salina cysts significantly decreased with increasing concentrations of the ethanolic extract and the positive control (potassium dichromate) while acetone and aqueous extracts had the same pattern of hatching with the highest hatching potential being observed at 0.125 mg/mL (Figure 8.2). The percentage hatching success of cysts incubated with the ethanol extract showed significant differences at varying

133

concentrations. The lowest concentration (0.0625 mg/mL) had the highest hatching percentage

(82%) and it was not significantly different from the cysts incubated at 0.125 mg/mL and 0.25 mg/mL with a hatching success of 67% and 60% respectively. There was zero percent (0%) hatchability observed from 0.25 mg/mL -1 mg/mL for potassium dichromate. The acetone, aqueous and ethanol extracts had significant higher hatching percentage of the cysts at 0.125 mg/mL (58%, 58% and 67% respectively). There was no significant difference in percentage hatching at 0.0625, 0.125, 0.25 and 1 mg/mL, while in the aqueous extract; also there was no significant difference in percentage hatching at 0.125, 0.25 and 1 mg/mL in the acetonic extract. In addition, no significant difference was observed in percentage hatching at 0.0625,

0.125, 0.25 and 0.5 mg/mL in the ethanolic extract and lastly there was no significant difference in percentage hatching at 0.0625, 0.125 and 0.25 mg/mL for the positive control (potassium dichromate) (p < 0.05

90 b 80 70 b a a,b b 60 a a b 50 a a 40 a a 30 a,c a c 20

10 c c c d 0 b

0.0625 mg/mL 0.125 mg/mL 0.25 mg/mL 0.5 mg/mL 1 mg/mL Hatchability success (%) success Hatchability

Aqueous extract Acetone extract Ethanol extract Potassium dichromate

Figure 8. 2: Percentage hatching success of A. salina cysts incubated in different solvent concentrations of K.africana and control. The values are means of the replicates (at different hours) for the concentrations for each plant extract/control ± SD of three replicates. Set of bars with different letters is significantly different (p < 0.05). 134

The effect of exposure time on the hatching success on A. Salina is shown in Figure 8.3. The same trend was observed in the acetone extract and sea water as there was significant differences in hatching success from 24 h to 72 h (p > 0.05). The lowest hatching success was observed at 24 hour in all the extracts and controls. The results from this study also showed that after 60 hours of exposure, hatching success of the cysts incubated in acetone and ethanol extracts only significantly increased by 1 and 1.1-fold, respectively while with aqueous decreased significantly by 1.2-fold (Figure 8.3). Potassium dichromate decreased significantly by 3.5-fold after 36 hours (p<0.05), followed by no hatching of cyst after 48 hours.

80 c a a a 70 a d a,b a a a a 60 a,b b a a a 50 b

40 a a,b 30 a,b 20 b c 10 c b c 0

Hatchability success (%) success Hatchability 24 h 36 h 48 h 60 h 72 h -10

Aqueous extract Acetone extract Ethanol extract Potassium dichromate Sea water

Figure 8. 3: Percentage hatching success of A. salina cysts incubated at different durations in K.africana extracts/controls. The values are means of replicates (of all the concentrations) for each plant extract/control ± SD of three replicates. Set of bars with different letters are significantly different (p < 0.05).

Brine shrimp lethality assay (BSLA)

The percentage lethality/mortality of A. salina larvae (nauplii) incubated in different solvent extracts of K. africana and controls are shown in Figure 4. There was a significantly higher

135

mortality percentage (99%) of the nauplii incubated with potassium dichromate than the extracts and sea water (p < 0.05).Although there was no significant difference (p < 0.05) between the aqueous and ethanolic extracts. The acetone extract had mortality of 18.20% while the sea water had the least mortality of 0%.

120

100 c

(%) 80

60 a a

40 Mortality b 20

d 0 Aqueous Acetone Ethanol Potassium Sea water extract extract extract dichromate

Figure 8. 4: Percentage mortality of A. salina nauplii incubated in different solvent extracts of K. africana and controls. Means are values of five concentrations for each plant fraction/control ± SD of three replicates. Bars with different letters are significantly different (p < 0.05).

The effect of varying concentrations of the plant fractions on the mortality of larvae is shown in Figures 8.5. The degree of mortality of nauplii was in a concentration-dependent fashion.

The highest mortality was observed in all the extracts at 1 mg/mL while the control had a maximum mortality (100%) at 0.125 mg/mL. There was no significant in percentage mortality of the nauplii at concentrations of 0.0625 - 1 mg/mL (p < 0.05) in the aqueous extract and potassium dichromate. There was also no significant in percentage mortality at concentrations of 0.125 - 1 mg/mL in the acetone extract while the ethanolic extract had no significant at

0.0625, 0.125 and 0.5 mg/mL (p < 0.05).

136

120

c c c 100 d c ac a

(%) 80 a 60 a a b 40 c a b Mortality b 20 a a,b b b b 0 0.0625 mg/mL 0.125 mg/mL 0.25 mg/mL 0.5 mg/mL 1 mg/mL

Aqueous extract Acetone extract Ethanol extract Potassium dichromate

Figure 8. 5: Percentage mortality of A. salina cysts incubated in different concentrations of the extracts of K.africana and control. The values are means of the replicates (at different hours) for the concentrations for each plant extract/control ± SD of three replicates. Set of bars with different letters are significantly different (p < 0.05).

The results also show that the mortality of nauplii incubated in these plant extracts increased with time (Figure 8.6). After 36, 48, 60 and 72 hours of incubation in acetone extract, the mortality significantly increased by 1.1, 1.1, 1.2 and 1.9-fold, respectively. With aqueous extract, the mortality significantly increased by 1.22, 1.5, 1.6 and 1.1 times more after 36, 48,

60 and 72 hours, respectively (p < 0.05). The nauplii death rate also increased by 1.13, 1.0, 1.8 and 1.1-fold after incubation in ethanol extract over the same period of time, respectively. The increase in mortality of the positive control at all duration times are 1.01, 1, 1 and 1-fold (p <

0.05). The sea water was observed to 0% mortality through the duration of the experiment. In overall, the mortality of nauplii was significantly similar when incubated with the ethanol, acetone extract and the aqueous which was significantly higher than in sea water (p < 0.05)

(Figure 8.6). 137

120 d 100 c c c c

80 a a a a 60 a 40 a c b b a Mortality(%) a 20 a b b b e d 0 d d 24 h 36 h 48 h 60 h 72 h Aqueous extract Acetone extract Ethanol extract Potassium dichromate Sea water

Figure 8. 6: Percentage mortality of A. salina cysts incubated in different time durations in extracts of K.africana /controls. The values are means of replicates (of all the concentrations) for each plant extract/control ± SD. Set of bars with different letters are significantly different (p < 0.05).

The estimated LD50 results were 1.11, 0.4498 and 0.298 mg/mL for the acetone and aqueous extracts, respectively (Table 1).

Table 8. 1. Lethal dose concentration (LD50) of acetone, ethanol and aqueous extracts of K. africana against Brine Shrimp

2 Sample Regression equation LD50 (mg/mL) Toxicity status R (%)

Aqueous extract Y = 25.247ln(x) + 80.6 0.298 Medium toxic 96.76

Acetone extract Y = 41.032x + 4.5 1.109 Non-toxic 96.21

Ethanol extract Y = 102.24x 0.489 Medium toxic 86.3

Potassium Y= 1.4427247ln(x) + 101 <0.100 Highly toxic 50 dichromate

The toxicity regression equation used to LD50 (mg/mL) values of acetone extract, aqueous extract, ethanol extract and potassium dichromate (positive control) lethality test in brine shrimp test. R2 (%) denotes the coefficient of determination of the regression equation

138

DISCUSSION In the last three decades, A. salina nauplii have been used as preliminary evaluation for general toxicity of herbal remedies. This present study evaluated the toxicity of K. africana using hatching success of cysts and mortality of the nauplii in different concentrations of plant extracts and controls. The hatching of A. salina cysts was highest overall in sea water (71%), while the ethanolic extract had the highest (49.2%) hatching success among the extracts used.

The ethanolic extract at 0.0625 mg/mL had the overall best hatching success. The hatching success significantly decreased with increasing concentrations of the plant extracts in a dose- dependent manner with potassium dichromate eliciting 100% hatching inhibition at 0.25 -1 mg/mL. This could be attributed to the high toxicity of potassium dichromate even at very low concentrations 21. A. salina is known to have a resistant cyst stage that can forbear a varied range of pH stretching from freshwater to saturated saline 20 and as such, if the dormancy is not broken hatching will not occur, therefore at 0.125mg/mL, the plant extracts exhibited an optimum breaking of dormancy of the cyst and further decrease in the concentration revealed an inhibitory action on the cyst.

Evaluation of the hatching success of the cysts in response to exposure time revealed that the aqueous and acetone extracts had no significant hatching success after 36 – 48 hours which is known to be the best hatching time for brine shrimp according to Meyer et al 15 whereas, cysts incubated in the ethanolic extract continued to hatch until the end of 72 hours. A moderate hatching success of the cysts was observed in all the extracts except for those incubated in potassium dichromate which had a hatching success as low as 4 % at 48 hours and hatching thereafter. The poor hatching success observed in potassium dichromate could be attributed to its toxic nature which could probably result to resistance of the eggs to hatching in response to chemical toxins.

139

The brine shrimp lethality results in this study were interpreted in accordance to Meyer’s toxicity index, LC50 < 1000 μg/mL (ppm) is toxic, while LC50 >1000 μg/mL is nontoxic.

Additionally, a more detailed criterion given by Clarkson as follows: LC50 >1000 μg/mL is nontoxic, LC50 500-1000 μg/mL is low toxic, LC50 100-500 μg/mL is medium toxic, and LC50

0-100 μg/mL is highly toxic 22 was used.

The results indicate that the aqueous and ethanolic solvent extracts of K.africana bulb exhibited moderate toxicity with LC50 of 298 and 489 μg/mL respectively; whereas the acetone extract were not toxic LC50 >1000 μg/mL. Hence, this extract may be considered safe for consumption as an herbal medicine.

On the other hand, its non-toxic nature discourages its use as an alternative for the treatment and management of cancer, whereas the aqueous and ethanolic extracts could serve in that regards 23.

Comparing the relationship between increase in concentration and lethality of the nauplii, we observed that the degree of mortality increased in a concentration dependent manner which peaked at 1 mg/mL. Only the acetonic extract was less toxic at 1 mg/mL with a mortality of

46%, whereas other fractions exhibited a mortality ranging from 84 - 100% at that same concentration. The mortality of nauplii incubated in these plant fractions increased exponentially with time, with the highest mortality observed at 72 hours for the plant extracts while in the case of potassium dichromate, maximum mortality was observed at 36 hours. The essence of exposing the nauplii to plant extracts over a long period of time was to determine their threshold of withstanding toxic metabolites/ chemical compounds present in the various

140

fractions. According to Carballo et al 24, maximum sensitivity of nauplii to test compounds is achieved at the second and third instar stage and it is interpreted to be after 48 hours of incubation. However, in this study it was not the case as maximum sensitivity was reached after 72 hours of exposure. This could be due to the presence of some nutritive metabolites that may have acted as food rather than toxic chemicals.

CONCLUSION The results of this study indicated that aqueous, acetonic and ethanolic extracts of Kedrostis africana bulbs supported hatching of cysts in the Brine Shrimp Assay. The acetonic extract was not toxic (LC50 > 1 mg/mL) whereas the both the aqueous and ethanolic displayed signs of moderate toxicity, suggesting the need for further in vivo and in vitro toxicological studies.

Cancer cell lines toxicity tests and isolation of cytotoxic compounds are necessary to ascertain if it has anticancer potentials. Based on the possible relationship between brine shrimp lethality and plant bioactivity, this work could serve for further pharmacological and phytochemical research.

141

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CHAPTER NINE

Toxicity evaluation of extracts from the combination of Kedrostis africana and Vernonia mespilifolia using brine shrimp model

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Chapter Nine Toxicity evaluation of extracts from the combination of Kedrostis africana and Vernonia mespilifolia using brine shrimp model

Contents Pages

Introduction ...... 147

Materials and methods ...... 147

Results and discussion ...... 149

Conclusion ...... 157

References ...... 158

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INTRODUCTION Medicinal plants and plant extracts are greatly relied upon by majority of the world population for the management, prevention and treatment of many aliments, most especially the noncommunicable and chronic ones (Rodriques and Casali, 2002). The assumption that medicinal plants are efficacious, natural, and their formulations are safe for the treatment of ailments could lead to indiscriminate use, most especially by traditional medicinal practitioners

(Ogbonnia et al. 2010).

Kedrostis africana and Vernonia mespilifolia belongs to the families Cucurbitaceae and

Asteraceae respectively. They are very important plants used in the Eastern Cape of South

Africa for the management or treatment of various ailments (Dolds and Cocks; George and

Nimmi, 2011). According to Afolayan and Mbaebie, (2010) the infusion from the combination of Kedrostis africana and Vernonia mespilifolia is used in traditional medicine for the management of obesity. Despite the documented use of the combination of these two plants for the management of obesity, to the best of our knowledge, there is a dearth of information regarding the safety in the short-term and long-term basis. The present study was designed to investigate the possible toxic effect of acetone, aqueous and ethanol extracts of the combination of Kedrostis africana and Vernonia mespilifolia on brine shrimp model.

MATERIALS AND METHODS Collection and extraction of sample were carried out as previously described in chapter five. A. salina hatching assay The method described by Otang et al. (2013) was employed with little modifications. Five petri dishes containing 30 mL of the extracts were prepared in filtered sea water by dissolving them in 1 mL of the parent solvents to yield a two-fold dilution of concentrations (1, 0.5, 0.25, 0.125 and 0.0625 mg/mL). A positive control was also prepared by dissolving potassium dichromate

147

in seawater in the same concentrations as the plant extracts; while sea water alone was used as the negative control. The setup was allowed to stand for 30 minutes to allow the solvents to evaporate.

Ten (10) A. salina cysts were stocked in each of the petri dishes containing 30 mL of the prepared two-fold concentrations (1 to 0.0625 mg/mL) of the plant fractions and positive control. The petri dishes were partly covered, incubated at 30°C under constant illumination for 72 hours. The number of free nauplii in each petri dish was counted after every 24 hours till the end of 72 hours. The percentage of hatchability was calculated by comparing the number of hatched nauplii with the total number of cysts stocked.

Artemia salina lethality assay Artemia salina cysts were hatched in sea water and 10 nauplii were pipetted into each petri dish containing the two-fold concentrations of the extracts and control as in the hatchability assay described above. The petri dishes were then examined and the number of living nauplii (that exhibited movement during several seconds of observation) was counted after every 24 hours and the set up was allowed to stand for 72 hours under constant illumination. The percentage of mortality (M %) was calculated as: Mortality (%) = (Total nauplii – Live nauplii) × 100 /

Total nauplii.

Data analysis

MINITAB version 17 for windows. One-way analysis of variance (ANOVA) followed by

RESULTS AND DISCUSSION Brine shrimp hatchability assay

The hatching success of Artemia salina incubated with different plant extracts and control is as shown in Figure 9.1 with the sea water having a significantly higher hatching success (71%) than the solvent extracts including the positive control (potassium dichromate) (5.4%) (p <

0.05). The hatching success of the cysts in the acetone (22.4%), aqueous (60.8%) and ethanol

(30.8%) extracts showed no significant difference from each other (p > 0.05). This could justify why most traditional herbal medicines are prepared using water as a solvent because it is not or less toxic. This could also bring to mind why the cyst showed more resistant to hatching in the acetonic and ethanol extracts than in the aqueous extracts. This resistant could be attributed to the permeability barrier provided by the cysts (Abu-Shanab et al. 2004).

149

80 e 70 a 60

40 c 30 b 20

10 d

Hatchability success (%) success Hatchability 0 Aqueous Acetone extract Ethanol extract Potassium Sea water extract dichromate

The effect of different solvent concentrations on the hatching success of the cyst was also evaluated and the result is depicted in Figures 9.2. Figure 9.2 shows the activities of the different plant extracts/positive control at varying concentrations to the hatching success of the cysts.

In the brine shrimp hatchability assay, the hatching success significantly decreased with increasing concentrations of the crude extracts in a dose dependent manner. The hatching success of the cyst was also evaluated at different concentrations and the result is depicted in

Figure 9.2. The aqueous extract exhibited its maximum hatching success at 0.125 mg/mL

(77%). The acetone and ethanol extracts had a similar hatching pattern, with the highest hatching success observed at 0.0625 mg/mL (Figure 9.2). The overall best percentage hatching success potential of cysts was observed with cyst incubated at 0.125 mg/mL in the aqueous extract which was significantly (P > 0.05) greater when compared with other solvent used.

150

Potassium dichromate showed the lowest hatching success. At concentrations of 0.0625 mg/mL and 0.125 mg/mL, there was no significant difference in the hatching success of acetone, aqueous and ethanol extracts. At concentrations of 0.25 mg/mL -1 mg/mL for potassium dichromate, no hatching was observed. Also, cysts incubated in 0.5 mg/mL and 1 mg/mL of acetone extracts did not hatch while those incubated in 1 mg/mL of ethanol extract did not hatch. The inability of cyst to hatch in the acetone and ethanol crude extracts of the combination of Vernonia mespilifolia and K. africana could be ascribed to the presence of toxic compounds in the extracts that have ovicidal property. In addition, it has been reported that secondary metabolites inherent plants could have effect on the embryonic development

(Subhadra et al. 2011).

90 a 80 a 70 a 60 a 50 a 40 c 30 b b 20 c 10 b

Hatchability success (%) successHatchability d b d b c d 0 0.0625 mg/mL 0.125 mg/mL 0.25 mg/mL 0.5 mg/mL 1 mg/mL Aqueous extract Acetone extract Ethanol extract Potassium dichromate

Figure 9.2: Percentage hatching success of A. salina cysts incubated in different concentrations of the extracts of combination of K. africana and V. mespilifolia and control. The values are means of the replicates (at different hours) for the concentrations for each plant extract/control ± SD of three replicates. Set of bars with different letters is significantly different (p < 0.05).

The results of the effect of exposure time on hatchability showed that the sensitivity of A. salina cyst to the plant extracts was strongly dependent upon exposure period (Figure 9.3). A similar

151

trend was observed with the aqueous extract and sea water as there was significant differences in hatching success from 24 h to 72 h (p > 0.05). The lowest hatching success was observed after 24 h treatment in all the extracts. This result is in line with report from Subhadra et al.

(2011) which stated that A. salina is extremely susceptible to toxin during its early stage of development. The acetone and ethanol extracts experienced maximum hatching at 60 h with

31% and 40% hatching success respectively, after which death set in for the already hatched nauphlii. The aqueous extract had the highest hatching success at 60 hours. Also in Figure 9.3 there was a 1.24 and 1.38 fold decrease respectively in hatched cyst from 36 to 60 h in both acetone and ethanol extracts while aqueous extract decreased significantly by 1.19 fold after

60 h (p < 0.05). Sea water exhibited optimum hatching at 36 h and remained the same throughout the experiment. Cysts incubated in potassium dichromate decreased significantly by 3.5-fold after 36 hours (p < 0.05), followed by no hatching of cyst after 48 hours.

80 a a a a a a a a 60 a

40 a b c c b b b b b

success (%) success 20 Hatchability b b b c d 0 b d 24 h 36 h 48 h 60 h 72 h Aqueous extract Acetone extract Ethanol extract Potassium dichromate Sea water

Figure 9.3: Percentage hatching success of A. salina cysts incubated at different durations in extracts of combination of K. africana and V. mespilifolia/controls. The values are means of replicates (of all the concentrations) for each plant extract/control ± SD of three replicates. Set of bars with different letters are significantly different (p < 0.05).

152

Brine shrimp lethality

The Brine shrimp lethality assay is considered as a preliminary assessment of toxicity. It determines the lethal concentration of active compounds such as heavy metals, pesticides, and medicines in brine medium (Piccardi et al. 2000) and it is being employed to determine toxicity of various active compounds because of it is reliable, rapid and very convenient to carry out

(Otang et al. 2013). The percentage mortality of A. salina larvae (nauplii) incubated in different solvent extracts of Vernonia mespilifolia and controls are shown in Figure 4. The aqueous extract has the highest mortality (68%) among all the extract tested. There was high mortality of nauplii incubated in both the acetone and ethanol extracts although there was no significant difference in their percentage mortality, whereas the nauplii incubated with potassium dichromate had a significantly greater mortality when compared to all the extracts and sea water (p < 0.05).

120

100 c

80 a

60 b

40 b Mortality (%) Mortality 20 d 0 Aqueous Acetone Ethanol extract Potassium Sea water extract extract dichromate

Figure 9. 4: Percentage mortality of A. salina nauplii incubated in different solvent extracts of combination of K. africana and V. mespilifolia plant extracts and controls. Means are values of five concentrations for each plant fraction/control ± SD of three replicates. Bars with different letters are significantly different (p < 0.05).

153

The effect of different concentrations of the plant extracts on the mortality of larvae is shown in Figures 9.5. The degree of mortality of nauplii was in a concentration-dependent fashion.

The highest mortality was observed in all the extracts at 1 mg/mL compared to potassium dichromate which showed maximum mortality (100%) at 0.125 mg/mL. There was no significant difference (p < 0.05) in percentage mortality of the nauplii between the acetone extract and ethanol extract at concentrations of 0.125 and 0.25 mg/mL. At 1 mg/mL, there was no significance difference in percentage mortality between aqueous and potassium dichromate

(p < 0.05) in Figure 9.5. The finding from this revealed that the effect of varying concentrations of all the plant extracts on the mortality of larvae was in a concentration dependent manner, thus it could be hypothesized from our findings that this plant possesses both toxicological and pharmacological potentials and as such it could be used interchangeable depending on the dosage administered (Otang et al. 2013; Kibiti and Afolayan, 2016).

120 d 100 d c c a a 80 a a a a b b 60 b c c 40 b b b

Mortality(%) c 20 b 0 0.0625 0.125 mg/mL 0.25 mg/mL 0.5 mg/mL 1 mg/mL mg/mL Aqueous extract Acetone extract Ethanol extract Potassium dichromate

Figure 9. 5: Percentage mortality of A. salina cysts incubated in different concentrations of the extracts of combination of K. africana and V. mespilifolia and control. The values are means of the replicates (at different hours) for the concentrations for each plant extract/control ± SD of three replicates. Set of bars with different letters is significantly different (p < 0.05).

154

The various extracts were screened at five different concentrations viz. 62.5, 125, 250, 500 and

1000 μg/mL, with their toxic effect observed on A. salina from 24 - 72 h. Potassium dichromate was used as a standard (Padmaja et al. 2002). The percentage mortality due to exposure time is as shown in Figure 9.6. The result revealed that the percentage mortality was time dependent; the longer the exposure of nauplii to the plant extracts, the greater the mortality. At 24 h, there was no significant difference in mortality of nauplii incubated aqueous, actone and ethanol extracts and as well as potassium dichromate. After 36, 48, 60 and 72 h of incubation in acetone extract, the mortality significantly increased by 3.43, 2.04, 1.24 and 1.20-fold, respectively.

With aqueous extract, the mortality significantly increased by 3, 1.33, 1.07 and 1.06 times more after 36, 48, 60 and 72 h, respectively (p < 0.05). The nauplii death rate also increased by 0, 0,

3.95 and 1.13-fold after incubation in ethanol extract over the same period of time, respectively.

The increase in mortality of the positive control at all duration times are 1.01, 1, 1 and 1-fold

(p < 0.05) Figure 9.6. The sea water was observed to 0% mortality through the duration of the experiment.

The nauplii attain the second and third instars of their life cycle within 48 hours hence reveal their greatest sensitivity to toxins at this time (Sreejamole and Radhakrishnan, 2013). However, the findings of this study indicate that the maximum sensitivity was reached after 72 h of exposure.

155

120

100 b d a a a a a a c c 80 b a b 60 b a 40 b c

20 Mortality(%) a c c d e 0 d 24 h 36 h 48 h 60 h 72 h -20

Aqueous extract Acetone extract Ethanol extract Potassium dichromate Sea water

Figure 9. 6: Percentage mortality of A. salina cysts incubated in different time durations in the plant extracts/controls. The values are means of replicates (of all the concentrations) for each plant extract/control ± SD. Set of bars with different letters are significantly different (p < 0.05).

According to Meyer et al. (1982) and Bastos et al. (2009) the brine shrimp lethality were interpreted in accordance to the criterion by Meyer toxicity index that LC50 values greater than

1000 μg/mL (1 mg/mL) are considered non-toxic, LC50 values equal/greater than 500 μg/mL

(0.5 mg/mL) but not greater than 1000 μg/mL are considered to have weak toxicity while those having LC50 values less than 500 μg/mL are considered toxic. The LC50 values were calculated as 20.96 µg/mL for aqueous extract, 354 µg/mL for acetone extract and 2264.97 µg/mL for the ethanol extracts, respectively (Table 9.1).

The brine shrimp lethality assay (BSLA) result revealed that the aqueous and acetone crude extracts of the combination of V. mespilifolia and K. africana showed that the extracts were toxic with LC50 <1 mg/mL (Table 9.1) hence these extracts may not be considered safe for consumption as herbal medicine. From the results, it could be deduced that the extracts could serve as a promising alternative in the treatment and management of tumour, as a brine shrimp

156

lethality test now serves as an indicator for the preliminary screening of bioactivity including for anticancer (Artanti et al. 2012).

Table 9. 1. Lethal dose concentration (LC50) of acetone, ethanol and aqueous extracts of combination of both V. mespilifolia and K. africana against Brine Shrimp

2 Sample Regression equation LC50 (µg/mL) Toxicity R (%) status Aqueous Y = 9.5218ln(x) + 86.8 20.96 Highly toxic 93.62 extract Acetone extract Y = 41.032x + 4.5 354 Medium toxic 88.21 Ethanol extract Y = 102.24x 2265 Non-toxic 86.82 Potassium Y= 1.4427247ln(x) + 101 <0.100 Highly toxic 50 dichromate

CONCLUSION The in vitro toxicity evaluation of the aqueous and acetone extracts of the combination of both plants against brine shrimp revealed that the combination of both plants may contain certain bioactive compounds which though toxic can be harnessed in the development of therapeutic drugs.

157

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obesity remedies in Nkonkobe Municipality of South Africa. Pharmacognosy

Magazine 2 (11), 368-373.

Artanti, N., Firmansyah, T., Darmawan, A., 2012. Bioactivities evaluation of Indonesian

mistletoe (Dendropthoe pentandra (L.) Miq.) leaves extracts. Journal of Applied

Pharmaceutical Science 02 (1), 2427.

Bastos, M.L.A., Lima, M.R.F., Conserva, L.M., Andrade, V.S., Rocha, E.M.M., Lemos,

R.P.L., 2009. Studies on the antimicrobial activity and brine shrimp toxicity of

Zeyheria tuberculosa (Vell.) Bur. (Bignoniaceae) extracts and their main

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Padmaja, R., Arun, P.C., Prashanth, D., Deepak, M., Amita, A., Anjana, M., 2002. Brine

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Subhadra, S., Kanacharalapalli, V.R., Rivindran, V.K., Parre, S.K., Chintala, S., Thatipally, R.,

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salina and animal cell lines. Journal of Natural Pharmaceuticals 3, 143-148.

160

CHAPTER TEN

Cytotoxicity evaluation of Kedrostis africana, Vernonia mespilifolia and the combination of both plants using HeLa cell line

161

Chapter Ten

Cytotoxicity evaluation of Kedrostis africana, Vernonia mespilifolia and the combination of both plants using HeLa cell line

Contents Pages

Introduction ...... 163

Materials and methods ...... 163

Results and discussion ...... 165

Conclusion ...... 171

References ...... 172

162

INTRODUCTION In South Africa, traditional medicine is a holistic form of healing which plays a vital role in the primary health care system. The native people of South Africa depend on herbal medicine for all aspects of primary health care. About fifteen million South Africans make use of traditional remedies from about seven hundred indigenous plant species (Campbell, 2007; Mander et al.

2007). According to Fyhrquist et al. (2002), more than 80% of the black populace do consult traditional healers for the management of myriad of ailments. There is often a widespread supposition that traditional remedies are non-toxic and safe because they are derived from natural sources (Fennell et al. 2004; Chen et al. 2011). Several reports have shown that traditional remedies are therapeutic at certain doses and toxic at another (Jha et al. 2003;

Boumba et al. 2004; Dwyer et al. 2005; Takeuchi et al. 2007).

The increasing growth in the use of medicinal plants for research and market demand for herbal medicines, brought forth the need for standardized and scientific evaluation with respect to their bioactive constituents and also the to determine their toxic constituents (Winston and

Maimes, 2007).

Cytotoxic assessment of plant extracts is frequently used as an initial phase of anti-obesity screening to assess the suitability of the extracts for in vitro cell-based assays. This cytotoxicity assay is based on cell density assays which evaluate the effects of the test samples on cell proliferation and/or cell death after exposure for specific time periods.

MATERIALS AND METHODS Collection and extraction of samples were carried out as previously described in chapter five but only the aqueous and ethanol extract were used for this study.

HeLa Cell culture HeLa (cervical cancer) cells were obtained from the laboratory of the Medicinal Plant and Natural

Product Research Group, Nelson Mandela Metropolitan University, South Africa). They were 163

routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum and seeded at a density of 6 000 cell/well in 96-well micro-titer plates with 200 μl of the medium.

Aqueous and ethanol extracts of V. mespilifolia, K. africana and their combination prepared in

DMSO (0.25%, v/v) were added to the wells already containing complete medium to reach concentrations of 50, 100 and 200 μg/ml of the extracts. The plates were then incubated for 48 hours at 37ºC in a humidified incubator and 5% CO2. Melphalan (100 μM) was used as positive control, while DMSO (0.25%, v/v) served as the vehicle (untreated) control.

In this study, the percentages of live and dead cells were analyzed using DNA-fluorescent dyes including Hoechst 33342 for total cell counts and Propidium iodide for dead cell counts. Cell imaging studies were performed using ImageXpress® Micro XLS widefield high-content analysis system. This is a widefield automated microscope capable of fluorescent, transmitted light and phase-contrast imaging and high-content analysis of fixed- or live-cell assays, tissues and small organisms.

Staining protocol: Hoechst 33342 and Propidium iodide staining Hoechst stock solution was prepared as a 10 mg/ml solution in distilled water. The stock solution was further diluted 1:2000 in PBS. All media were removed from the cultured cells and replaced with 100 μl of the Hoechst solution. The plates were incubated for about 20 minutes at room temperature away from light using an aluminum foil paper. Propidium iodide

(PI) (50 μg/ml) was then added shortly before acquiring the images. Note that PI was added very close to acquiring images to prevent prolonged contact of the dye with the cells, as prolonged contact causes the entry of PI into live cells.

Image acquisition and data analysis Images were taken with a Molecular Devices ImageXpress Micro XLS microscope using the blue and red filters, as well as phase contrast with 40X objective. Nine fields were acquired per well cells were scored by defined dimensions and analyzed by the MetaXpress software. 164

RESULTS AND DISCUSSION Evaluation of cytotoxicity against HeLa cells The present study assessed the possibility of cytotoxicity in HeLa cells following exposure to aqueous and ethanol extracts of V. mespilifolia, K. africana and their combination. Images acquired after Hoecsht 33342 and propidium iodide staining are presented in Figure 10.1, 10.2 and 10.3. Hoechst® 33342 nucleic acid stain is a popular cell-permeant nuclear counterstain that emits blue fluorescence when bound to dsDNA, while Propidium iodide (PI) is a popular red-fluorescent nuclear and chromosome counterstain (Belloc et al. 1994; Scientific, 2016;

Sigma-Aldrich, 2016). As propidium iodide is not permanent to live cells, it is also commonly used to detect dead cells in a population. PI binds to DNA by intercalating between the bases with little or no sequence preference. These conditions allow the identification of both cytotoxic and cytostatic compounds, while at the same time minimise potential problems such as nutrient deprivation which may occur with longer drug exposure time and higher cell densities. It was observed that the ethanol extracts of all the plants used were cytotoxic to HeLa cells while the aqueous extracts at the highest concentration were cytostatic (i.e. it inhibits cell growth and multiplication) to HeLa cells. Hence, it is therefore important to identify compounds that induce cytotoxicity in cancer cells as this is an important initial step in the development of anti-cancer drugs. These compounds need to kill cancer cells at a concentration that does preferably not harm normal cells (Lupi and Del Prato, 2008).

165

166

Figure 10. 2: Hoechst (blue) and Propidium iodide (red) staining of HeLa cells following 48 hours exposure to aqueous and ethanol extracts K. africana at 200 μg/mL and the positive control (Melphalan; 100 μg/mL). Blue staining indicates live cells; red staining indicates dead cells. Where a= untreated control group; b= positive control (Melphalan; 100 μg/mL); c= aqueous extract at 100 μg/mL and d=ethanol extract at 100 μg/mL.

167

Figure 10. 3: Hoechst (blue) and Propidium iodide (red) staining of HeLa cells following 48 hours exposure to aqueous and ethanol extracts of the combination of both plants at 200 μg/mL respectively and the positive control (Melphalan; 100 μg/mL). Blue staining indicates live cells; red staining indicates dead cells. Where a= untreated control group; b= positive control (Melphalan; 100 μg/mL); c= aqueous extract at 100 μg/mL and d=ethanol extract at 100 μg/mL.

168

After 48 hrs of exposure to the extracts, HOE/PI assay in HeLa cells revealed the pattern of cytotoxicity as shown in Figure 10.4. In the aqueous extracts, the percentage of dead cells obtained upon the 48 hrs exposure of V. mespilifolia were 2.31, 1.40 and 3.79% at doses 50,

100 and 200 μg/mL respectively while that of K. africana was 0.64, 1.45 and 2.17% at 50, 100 and 200 μg/mL, respectively, also in the combination of both plants 0.76, 0.82 and 1.37% of dead cells were obtained at doses of 50, 100 and 200 μg/mL respectively. All these values were significantly lower (p < 0.05) than that of the positive control (melphalan) (38.58%) at 100

μg/mL (Figure 10.4a).

The ethanolic extracts showed almost the same trend of dose dependent pattern of cytotoxicity.

The percentage of dead cells in V. mespilifolia produced a higher number of percentage dead cells compared to aqueous extract with values of 5.48 and 27.15% at 50 and 100 μg/mL which were significantly lower (p < 0.05) than that of the positive control (38.58%) at 100 μg/mL but

73.24% at 200 μg/ml, which were significantly higher (p < 0.05) than that of the positive control (38.58%) at 100 μg/mL (Figure 10.2 b). K. africana produced a percentage dead cells of 19.88, 15.41 and 7.87% at doses of 50, 100 and 200 μg/ml, respectively which were significantly lower (p < 0.05) than that of the positive control (melphalan) (38.58%) at 100

μg/mL while the combination of both plants revealed values of 5.48% at 50 μg/mL which were significantly lower (p < 0.05) than that of the positive control (38.58%) at 100 μg/mL but 41.12 and 73.24% at 100 and 200 μg/ml, which were significantly higher (p < 0.05) than that of the positive control (38.58%) at 100 μg/mL(Figure 10.4b).

169

45 Cytotoxicity of aqueous extracts in HeLa cells 40 a 35 30 25 20

15 a 10 b

Cell death (% of control) of (% death Cell 5 b c b b a c 0 Control 50 μg/mL 100 μg/mL 200 μg/mL Control Melphalan V. mespilifolia K. africana Combination

100 Cytotoxicity of ethanol extracts in HeLa cells 90 c 80 b 70 60 50 d 40 a c 30 b 20 b a d 10 c a

0 % Cell death (% of control) of (% death Cell % Control 50 μg/mL 100 μg/mL 200 μg/mL Control Mel EKA EVM EC

Figure 10. 4: HOE/PI cytotoxicity (expressed as % of control ± standard deviation; n = 3) of a) aqueous extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. b) ethanol extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. Values are mean ± SD (n = 3). Mean separation by LSD (p < 0.05). Set of bars (the same concentration) with different alphabets are different.

170

Table 10.1 shows the results of the preliminary screening of the crude extracts of V. mespilifolia, K. africana and their combination. A complete dose-dependent curves were generated and IC50 values were calculated for these crude extracts, averaged from 3 experiments, against HeLa (cervical cancer). According to the United States America National

Cancer Institute of Plant Screening Program, any plant extract with an IC50 value equal to or lesser than 20 휇g/mL after an incubation period 48 hrs is generally considered to have active cytotoxic effect (Geran et al. 1972; Lee and Houghton, 2005; Malek et al. 2009; Vijayarathna and Sasidharan, 2012). From the above statement, it can be deduced that all the extracts screened using HeLa cells are not cytotoxic because their IC50 values are far greater than 20

휇g/mL. These relatively low toxicity result of all the extracts used for this study suggests that these extracts may pose relatively low risks in their use for anti-obesity treatment.

Table 10. 1. IC50 values (µg/ml) of plant extract treatment of HeLa cells after 48 hours Treatment V. mespilifolia K. africana Combination of both plants Aqueous >200 >200 >200 Ethanol 149.73 >200 121.73

CONCLUSION The cytotoxic effect of the aqueous and ethanol extracts of V. mespilifolia, K. africana and their combination against HeLa cells, showed that all the extracts tested were not cytotoxic to the HeLa cells and thus make them suitable in the management of obesity.

171

REFERENCES Belloc, F., Dumain, P., Boisseau, M.R., Jalloustre, C., Reiffers, J., Bernard, P., Lacombe, F.,

1994. A Flow Cytometric Method Using Hoechst 33342 and Propidium Iodide

for Simultaneous Cell Cycle Analysis and Apoptosis Determination in Unfixed

Cells. Cytometry 17:59-65.

Boumba, V.A., Mitselou, A., Vougiouklakis, T., 2004. Fatal poisoning from ingestion of

Datura stramonium seeds. Veterinary and Human Toxicology 46, 81–82.

Campbell, C. 2007. Traditional medicine supplies falling as demand rises. Science and

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traditionally to treat cancer. Journal of Ethnopharmacology 100 (3), 237–243.

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consequence. Diabetes & Metab. 34, S56–S64.

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Pereskia bleo (Kunth) DC. (Cactaceae) leaves. Molecules 14 (5), 1713–1724.

Mander, M., Ntuli, L., Diederichs, N., Mavundla, K., 2007. Economics of the traditional

medicine trade in South Africa. White River Jet., VT: Chelsea Green.

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174

CHAPTER ELEVEN

In vivo toxicological evaluation of aqueous extracts of Kedrostis africana, Vernonia mespilifolia and the combination of both plants using Wistar rat model

175

Chapter Eleven

In vivo toxicological evaluation of aqueous extracts of Kedrostis africana, Vernonia mespilifolia and the combination of plants using Wistar rat model

Contents Pages

Introduction ...... 177 Materials And Method ...... 178 Results And Discussion ...... 182 Conclusion ...... 201 References ...... 202

176

INTRODUCTION There is a global increase in the consumption of herbal formulations by the public owing to the strong belief that these products are natural and hence are thought to be safe for the treatment of ailments (Said et al. 2002). However, there is little information or evidence available regarding the possible toxicity of herbal remedies and the adverse effect they may cause to the consumer (Ping et al. 2013). Consumers are primarily interested in fast access to safe, cheap, easily accessible and efficient herbal remedies while their potential toxic effects resulting from their short-term and long-term use are not taken into consideration as their toxic effect cause damage to internal organs (Ogbonnia et al. 2009).

Hence, findings from acute and subacute toxicity studies on herbal remedies should be obtained in order to increase the confidence in their safety to humans, particularly for use in the development of pharmaceutical (Ukwuani et al. 2012). It is therefore important to evaluate the toxicological effects of any medicinal plant extract intended to be used in animal or human to ascertain its potential toxic effects.

Despite the use of V. mespilifolia, K. africana and their combination traditionally,there is no report on the toxicity of any part of the plant in vivo. The scientific information regarding the safety of the use of this plant as an alternative medicine is therefore very important before it can be further developed into a new medicinal herbal therapy. In view of this, the objective of this study was to investigate the acute and subacute toxicity of V. mespilifolia, K. africana and their combination in Wistar rats.

177

MATERIALS AND METHOD Collection and extraction of sample were carried out as previously described in chapter five but only the aqueous extracts were used in this study.

Experimental animals

Healthy albino rats of the Wistar strain (both sexes), purchased from the South African Vaccine

Producers, Johannesburg, South Africa were used for the studies. They were housed in the animal house of the Central Analytical Laboratory, University of Fort Hare Alice, 5700, South

Africa . The rats were kept under standard laboratory conditions (12 h light and dark cycle; 22

± 2 °C). The animals were fed with standard rat pellet diet and water ad libitum. The study was carried out and the animals were handled according to the guidelines of the National Research

Council: Guide for the Care and Use of Laboratory Animals, Committee on Care and Use of

Laboratory Animals. Institute of Laboratory Animal Resources DHHS (NIH Publication No.

1985:85–93). The study was approved by the University of Fort Hare Animal Use Research

Ethics Committee, South Africa with protocol number AFO051SUNU01.

Acute oral toxicity study

The acute toxicity study was conducted following the OECD guidelines 420 (OECD, 2001).

Healthy female and male albino rats of the Wistar strain weighing between 95 and 120 g were used. A single of 2000 and 5000 mg/kg body weight was administered to the animals. Animals were randomly divided into 14 groups (3 females and 3 males) of 3 animals each. Groups 1 and 2 were respectively female and male controls. Groups 3-8 were experimental female and male rats respectively, and they were given aqueous extracts of V. mespilifolia, K. africana and their combination at a dose of 2000 mg/kg body weight while groups 9-14 were also experimental female and male rats respectively, both were given 5000 mg/kg body weight

(OECD, 2003). The animals were maintained on a standard animal diet and water. The extract

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when applied was suspended in a vehicle (distilled water) and administered orally by gavage following an overnight fast but with access to water. All animals were observed for general behavioral changes; symptoms of toxicity, changes in body weight and mortality after treatment for the first 4 h, then over a period of 24 h, thereafter daily for 14 days. The rats were fasted for 16 - 18 h and then sacrificed rats were anaesthetized using halothane and blood samples were collected via cardiac puncture into heparinized and EDTA containing bottles and subsequently used for biochemical and hematological analysis. The liver, kidney, heart, lung and pancreas were excised, weighed and observed for any histopathological defects.

Subacute oral toxicity study

Subacute oral toxicity was conducted in accordance with the Organization for Economic

Cooperation and Development (OECD) Test Guidelines 407 with slight modifications (OECD,

2008).

Animal grouping:

One hundred healthy animals were randomly divided into the following 20 groups; each group consisting of five animals. Animal grouping and their treatment were as follows:

Groups- I and II: Control (female and male)

Groups -III and IV: aqueous extract of K. africana at 200 mg/kg (female and male)

Groups- V and VI: aqueous extract of K. africana at 400 mg/kg (female and male)

Groups -VII and VIII: aqueous extract of K. africana at 600 mg/kg (female and male)

Groups -IX and X: aqueous extract of V. mespilifolia at 200 mg/kg (female and male)

Groups XI and XII- : aqueous extract of V. mespilifolia at 400 mg/kg (female and male)

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Groups- XIII and XIV: aqueous extract of V. mespilifolia at 600 mg/kg (female and male)

Groups- XV and XVI: aqueous extract of the combination of both plants at 200 mg/kg (female and male)

Groups- XVII and XVIII: aqueous extract of the combination of both plants at 400 mg/kg

(female and male)

Group- XIX and XX: aqueous extract of the combination of both plants at 600 mg/kg (female and male).

The aqueous extracts of V. mespilifolia, K. africana and the combination of both plants was rally administered to each group of rats daily for 28 days, while the control group received the distilled water as vehicle. Body weights of the rats in all groups were recorded before the start of dosing once and weekly during the treatment period and finally on the days of sacrifice.

Food and water in-take were recorded daily. Rats were fasted overnight, anesthetized using diethyl ether and sacrificed after the 29th day. Paired blood samples, heparinised and non- heparinised, were collected for hematological and serum biochemical assays.

Relative organ weight

On the 29th day, the animals were humanely sacrificed following an overnight fast. Organs such as liver, lungs, kidneys, heart and spleen were carefully dissected out and weighed in grams. The relative organ weight of each animal was calculated as follows:

Relative organ weight = absolute organ weight (g) X 100/ body weight of rats on sacrifice day

(g)

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Blood sampling

Blood samples were collected via cardiac puncture technique. Blood was divided into two parts; one part was collected in plain bottles (non-heparinized), while the second part was collected in heparinized bottles. The blood samples were subsequently centrifuged at 3000 rpm for 10 min using a bench centrifuge (Hermle Z 306, Labortechnik GmbH, Germany) to obtain serum and plasma respectively. The serum and plasma obtained was separated, and transferred into fresh plain sample bottles and used for the subsequent haematological and biochemical analyses.

Hematological analysis

The heparinized blood was analysed for white blood cell (WBC) count, red blood cell (RBC) count, differential leukocyte counts, red cell distribution width (RCDW), platelets count, haematocrit, hemoglobin estimation (HB), mean cell volume (MCV), mean corpuscular hemoglobin (MCH) mean corpuscular hemoglobin concentration (MCHC), Neutrophils,

Lymphocytes, Monocytes, and Eosinophils (Obici et al. 2008; Tan et al. 2008).

Biochemical analysis

Biochemical analysis carried out include measurement of activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and gamma-glutamyl transferase (ƔGT) activities. Other assays include total protein, calcium, magnesium, chloride, glucose and albumin to assess the liver function; serum total protein, urea, uric acid, creatinine, total bilirubin (Total bil) and conjugated bilirubin (Con bil) concentrations for kidney function. Also, the concentration of serum triglyceride and total cholesterol were also determined to give an indication of the effects of these extracts on lipid profile.

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Histopathological studies

The liver and kidney excised from each group of the animals were subjected to histopathological examinations. After fixing the tissues in 10% formalin, they were dehydrated and mounted in paraffin blocks. Sectioning was done at 5-7 μM. Routine histopathology was performed using the Haemotoxylin stain.

Data analysis

The statistical analysis was done using MINITAB version 17 for Windows. Data are expressed as mean ± standard deviation (SD, n = 5) and were subjected to oneway analysis of variance

(ANOVA) followed by Fischer’s Least Significant Difference to determine significant difference for all the parameters. Values were considered statistically significant at p < 0.05.

RESULTS AND DISCUSSION Acute toxicity At graded doses of 2000 and 5000 mg/kg body weight of the administration of aqueous extracts of V. mespilifolia, K. africana and their combination, the animals did not show any signs of adverse reaction and no visible changes in behaviours. There was no significant difference in water and food intake and also weight gain in both male and female rats during daily monitoring up the 14th day after the administration of the extracts. In addition, no mortality was recorded throughout the period of observation. As there was no mortality and clinical signs of toxicity in all the tested doses, LD50 value of the aqueous extracts was found to be greater than 5000 mg/kg. Any pharmaceutical drug or compound with an oral LD50 higher than 1000 mg/kg could be considered safe and of low toxic (Adeneye et al. 2009) (data not shown).

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Subacute toxicity

Effect of aqueous extracts of V. mespilifolia, K. africana and the combination of both plants on food intake and water consumption in rats

During dosing (28-days) there was no significant difference (p>0.05) change in food and water intake in both female and male rats at the doses of V. mespilifolia, K. africana and the combination of both plants tested compared to their respective control groups (Table 11.1 a, b and c). This is an indication that the animals were having healthy growth based on their food intake as well as the plant extract. Reports have it that animals that lose 10% of the initial body weight might not survive, which is an indication of adverse side effects of phytomedicines.

(Feres et al. 2006; Wonder et al. 2011).

Treatment Sex Average food intake Average water intake (g/day/rat) (mL/day/rat) Control Female 12.73 + 1.64a 18.74 + 3.94a

Male 18.34 + 1.39a 24.19 + 4.96a 200 mg/kg Female 11.60 + 0.50a 16.81 + 1.97a

Male 17.92 +0.98a 22.21 + 3.09a 400 mg/kg Female 12.21 + 1.16a 17.11 + 2.29a

Male 17.91 + 1.00a 24.91 + 5.79a 600 mg/kg Female 12.08 + 0.97a 15.39 + 0.59a

Male 17.90 + 0.96a 24.64 + 7.49a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

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Treatment Sex Average food intake Average water intake (g/day/rat) (mL/day/rat) Control Female 12.73 + 1.64a 18.74 + 3.94a

Male 18.34 + 1.39a 24.19 + 4.86a

200 mg/kg Female 12.09 + 0.99a 16.07 + 1.26a

Male 16.94 + 0.01a 21.71 + 2.59a

400 mg/kg Female 11.55 + 0.45a 16.32 + 1.48a

Male 16.97 + 0.03a 22.39 + 3.29a

600 mg/kg Female 11.73 + 0.65a 17.28 + 2.51a

Male 16.96 + 0.02a 22.12 + 2.98a

Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05). c)

Treatment Sex Average food intake Average water intake (g/day/rat) (mL/day/rat) Control Female 12.73 + 1.64a 18.74 + 3.94a

Male 18.34 + 1.39a 24.19 + 4.86a 200 mg/kg Female 12.54 + 1.44a 19.65 + 4.84a

Male 17.56 + 0.64a 25.17 + 6.02a 400 mg/kg Female 12.66 + 1.56a 18.24 + 3.48a

Male 18.07 + 1.14a 25.67 + 6.52a 600 mg/kg Female 11.32 + 0.22a 16.06 + 1.28a

Male 17.46 + 0.49a 24.36 + 5.24a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

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Effects of aqueous extracts of V. mespilifolia, K. africana and the combination of both

plants on body weights

Oral administration of aqueous extracts of V. mespilifolia, K. africana and the combination of

both plants at doses of 200, 400 and 600 mg/kg body weight for 28 days did not produce any

mortality in tested animals. No sign of observable toxicity was detected during the experiment.

There was no significant difference in the body weights of V. mespilifolia, K. africana and the

combination of both plants treated rats in comparison with the control groups (Table 11.2).

Decrease or increase in body weight is associated with toxic effects of chemicals and drugs.

Research has shown that increase or decrease in body weights are associated with the

accumulation of fats or physiological adaptive responses to xenobiotic which leads to reduced

appetite leading to low caloric intake by the animals (Arsad et al. 2013).

Table 11. 2. Body weights(g) of female and male rats following 28-days subacute oral administration of different doses of a) V. mespilifolia, b) K. africana, and c) combination of both plants at different dose.

Body weight (g/week) Female 0 1 2 3 4 Control 102.05±7.08a 129.99±9.50a 163.32±7.41a 169.60±9.89a 187.03±6.96a 200 mg/kg 99.92±4.46a 123.18±2.75a 158.73±2.70a 164.53±4.78a 187.46±7.39a 400 mg/kg 95.98±0.98a 121.59±1.21a 158.03±2.10a 168.56±8.81a 185.25±5.18a 600 mg/kg 96.98±1.81a 124.71±4.39a 159.18±3.23a 167.63±7.82a 186.24±6.12a Male Control 114.12±6.47a 152.47±11.94a 213.40±23.39a 241.73±12.79a 281.10±10.53a 200 mg/kg 109.70±3.07a 149.57±9.06a 194.16±4.23a 238.78±9.87a 276.49±5.96a 400 mg/kg 107.38±0.74a 147.66±6.98a 197.21±7.26a 237.44±8.58a 273.25±2.71a 600 mg/kg 110.32±3.68a 144.37±3.84a 199.45±9.49a 236.06±7.17a 278.58±8.03a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

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Body weight (g/week) Female 0 1 2 3 4 Control 102.05±7.08a 129.99±9.50a 163.32±7.41a 169.60±9.89a 187.03±6.96a 200 mg/kg 97.77± 2.78a 124.59±4.29a 162.85±6.91a 169.50±9.75a 185.29±5.25a 400 mg/kg 99.26± 4.29a 120.76±0.38a 159.40±3.46a 166.46±6.69a 185.82±5.74a 600 mg/kg 105.67±10.67a 125.88±5.49a 156.05±0.12a 164.12±4.37a 184.05±3.96a Male Control 114.12±6.47a 152.47±11.94a 213.40±23.39a 241.73±12.79a 281.10±10.53a 200 mg/kg 114.51±7.46a 148.87±8.34a 202.64±12.71a 238.73±9.82a 275.59±5.08a 400 mg/kg 108.49±1.83a 143.64±3.11a 209.62±19.67a 240.53±11.67a 280.43±9.85a 600 mg/kg 112.17±5.49a 148.82±8.28a 206.06±16.12a 239.40±10.51a 278.19±7.65a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05). c)

Body weight (g/week) Female 0 1 2 3 4 Control 102.05±7.08a 129.99±9.50a 162.32±7.41a 169.60±9.89a 187.03±6.96a 200 mg/kg 99.45±4.45a 126.34±5.95a 159.18±3.27a 168.17±8.42a 183.17±3.10a 400 mg/kg 96.83±1.82a 128.66±8.26a 157.44±1.52a 169.21±9.43a 186.21±7.13a 600 mg/kg 109.94±19.92a 127.64±7. 28a 156.08±0.13a 166.12±6.34a 185.12±5.04a Male Control 114.12±6.47a 152.47±11.94a 213.40±23.39a 241.73±12.79a 281.10±10.53a 200 mg/kg 119.02±12.36a 154.73±10.20a 229.01±39.08a 246.76±17.86a 279.76±9.26a 400 mg/kg 114.57±7.91a 163.88±23.32a 217.38±27.45a 249.31±20.84a 281.31±10.77a 600 mg/kg 112.83±6.18a 169.98±29.47a 223.01±33.03a 252.71±23.79a 276.51±5.99a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

Effect of aqueous extracts of V. mespilifolia, K. africana and the combination of both

plants on organ weight

There was no significant difference (p>0.05) in organ weights of rat treated with aqueous

extracts of V. mespilifolia, K. africana and the combination of both plants as compared to the

control rats after 28-days treatment (Table 11.3a, b and c). Changes in organ weights is one of

the indices used for determining toxicity in animals. There is a very high likelihood that the

ingestion of herbal products into the body may be toxic to vital organs, such as the kidney,

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liver, spleen, heart and brain because of their diverse roles in the human body (Ndhlala et al.

2013). As a result there no significant increase in organ weight it can be deduced that the aqueous extract of the plants were not toxic.

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Table 11. 3. Relative organ weights (per 100g body weight) of rats in subacute toxicity of aqueous extracts of a) V. mespilifolia, b) K. africana and c) combination of both plants. a)

Relative Organ weight (g) Group Male Female

Heart Liver Lungs Kidney Spleen Heart Liver Lungs Kidney Spleen Control 0.96 ± 9.11 ± 1.11 ± 2.02 ± 0.60 ± 0.61 ± 6.04 ± 1.01 ± 1.33 ± 0.47 ± 0.03a 3.34a 0.39a 0.72a 0.21a 0.33a 3.23a 0.54a 0.71a 0.25a 200 mg/kg 0.92 ± 9.05 ± 1.09 ± 2.01 ± 0.60 ± 0.61 ± 6.01 ± 1.19 ± 1.21 ± 0.46 ± 0.00a 3.27a 0.42a 0.74a 0.23a 0.36a 2.94a 0.70a 0.55a 0.26a 400 mg/kg 0.93 ± 9.09 ± 1.15 ± 2.06 ± 0.6 ± 0.24a 0.62 ± 6.10 ± 1.12 ± 1.22 ± 0.46 ± 0.01a 3.32a 0.41a 0.78a 0.37a 3.08a 0.67a 0.57a 0.24a 600 mg/kg 0.91 ± 9.08 ± 1.09 ± 2.05 ± 0.61 ± 0.64 ± 6.07 ± 1.15 ± 1.25 ± 0.45 ± 0.00a 3.31a 0.43a 0.77a 0.24a 0.37a 3.14a 0.68a 0.62a 0.23a

Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

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b) Relative Organ weight (g)

Group Male Female

Heart Liver Lungs Kidney Spleen Heart Liver Lungs Kidney Spleen Control 0.96 ± 0.03a 9.11 ± 3.34a 1.11 ± 0.39a 2.02 ± 0.72a 0.60 ± 0.21a 0.61 ± 0.33a 6.04 ± 3.23a 1.01 ± 0.54a 1.35 ± 0.71a 0.47 ± 0.25a 200 mg/kg 0.96 ± 0.05a 9.08 ± 3.32a 1.12 ± 0.36a 2.08 ± 0.79a 0.62 ± 0.24a 0.6 ± 0.36a 6.03 ± 3.13a 1.04 ± 0.56a 1.37 ± 0.70a 0.47 ± 0.26a 400 mg/kg 0.90 ± 0.00a 9.09 ± 3.27a 1.13 ± 0.39a 2.02 ± 0.71a 0.59 ± 0.21a 0.60 ± 0.36a 5.63 ± 3.17a 1.02 ± 0.52a 1.36 ± 0.71a 0.48 ± 0.25a 600 mg/kg 0.98 ± 0.06a 9.12 ± 3.33a 1.14 ± 0.41a 2.07 ± 0.76a 0.59 ± 0.21a 0.59 ± 0.33a 5.36 ± 3.04a 1.05 ± 0.59a 1.36 ± 0.72a 0.46 ± 0.26a

Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

Relative Organ weight (g)

Groups Male Female Heart Liver Lungs Kidney Spleen Heart Liver Lungs Kidney Spleen Control 0.96 ± 0.05a 9.11 ± 3.34a 1.11 ± 0.39a 2.02 ± 0.72a 0.60 ± 0.21a 0.61 ± 0.33a 6.04 ± 3.23a 1.01 ± 0.54a 1.33 ± 0.71a 0.47 ± 0.25a 200 mg/kg 0.96 ± 0.07a 9.12 ± 3.35a 1.13 ± 0.42a 2.09 ± 0.77a 0.62 ± 0.24a 0.64 ± 0.35a 6.03 ± 3.27a 1.03 ± 0.57a 1.37 ± 0.75a 0.43 ± 0.21a 400 mg/kg 1.00 ± 0.09a 9.12 ± 3.34a 1.18 ± 0.46a 2.07 ± 0.75a 0.62 ± 0.26a 0.61 ± 0.32a 6.04 ± 3.24a 1.02 ± 0.52a 1.33 ± 0.69a 0.40 ± 0.19a 600 mg/kg 1.06 ± 0.17a 9.12 ± 3.37a 1.13 ± 0.41a 2.08 ± 0.79a 0.62 ± 0.23a 0.68 ± 0.39a 6.07 ± 3.31a 1.04 ± 0.54a 1.34 ± 0.75a 0.46 ± 0.26a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05). 189

Effect of the aqueous extracts of V. mespilifolia, K. africana and the combination of both plants on haematological parameters of rats following a 28 days.

The effect of daily administration of the aqueous extracts of V. mespilifolia, K. africana and the combination of both plants on different haematological parameters namely WBC, RBC,

Hb, MCV, MCH, MCHC, RDW, MPV platelets, neutrophil, lymphocyte, basophils, haematocrit, eosinophil and monocyte did not show any significant difference (p>0.05) between the experimental and control groups at all the tested doses for both sexes (Tables 11.4,

11.5 and 11.6).

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Table 11. 4. Haematological values of rats in subacute toxicity of V. mespilifolia

Parameter Male Female Control 200 mg/Kg 400 mg/Kg 600 mg/Kg Control 200 mg/Kg 400 mg/Kg 600 mg/Kg WBC (X 109/L) 10.14 ± 8.65 ± 2.06a 8.33 ± 1.87a 8.36 ± 1.92a 7.42 ± 1.65a 8.89 ± 3.13a 8.57 ± 2.80a 9.43 ± 3.68a 3.55a RBC (X 1012/L) 7.74 ± 0.30a 7.88 ± 0.33a 8.06 ± 0.62a 8.17 ± 0.72a 8.15 ± 0.14a 8.22 ± 0.29a 11.78 ± 8.53 ± 0.78a 4.03a Haem (g/dL) 14.97 ± 15 ± 0.43a 15.2 ± 0.24a 15.2 ± 0.37a 14.97 ± 15.1 ± 0.14a 15.73 ± 15.33 ± 0.31a 0.09a 0.72a 0.41a MCV (fL) 64.93 ± 64.37 ± 64.23 ± 64.50 ± 63 ± 3.24a 60.5 ± 0.79b 62.17 ± 61.57 ± 0.90a 0.28a 0.14a 0.30a 2.49a 1.79a MCH (pg) 19.3 ± 0.29a 19.03 ± 18.93 ± 19.77 ± 18.43 ± 18.37 ± 18.37 ± 18.33 ± 0.29a 0.04a 0.83a 0.29a 0.26a 0.28a 0.26a MCHC (g/dL) 29.73 ± 29.6 ± 0.25a 29.3 ± 0.22a 29.13 ± 29.23 ± 30.43 ± 30.07 ± 29.2 ± 0.09a 0.26a 0.26a 0.26a 1.24a 0.92a RDW (%) 11.5 ± 0.14a 11.63 ± 11.60 ± 11.5 ± 0.14a 10.87 ± 10.2 ± 0.14a 10.63 ± 10.67 ± 0.33a 0.33ab 0.81a 0.53a 0.56a PC (X 109/ L) 781 ± 65.31a 770 ± 52.81a 774 ± 55.45a 771.67 ± 778.67 ± 773.33 ± 774 ± 94.78a 777.67 ± 50.65a 92.32a 91.28a 94.67a MPV Fl 9.97 ± 0.05a 9.93 ± 0.05a 9.98 ± 0.07a 9.94 ± 0.09a 9.27 ± 0.31a 9.5 ± 0.52a 9.07 ± 0.06a 9.57 ± 0.52a Neutrophils (X 0.41 ± 0.10a 0.87 ± 0.56a 0.6 ± 0.37a 0.51 ± 0.27a 0.64 ± 0.06a 0.82 ± 0.27a 1.08 ± 0.47a 0.62 ± 0.05a 109/L) Lymphocytes( X 6.32 ±0.90a 6.48 ± 0.96a 6.43 ± 0.93a 6.46 ± 0.97a 4.84 ± 0.94a 5.82 ± 1.90a 5.36 ± 1.49a 6.16 ± 2.23a 109/L) Monocytes (X 109/L) 2.09 ± 0.12a 2.07 ± 0.16a 2.05 ± 0.13a 2.03 ± 0.14a 1.34 ± 0.53a 1.39 ± 052a 1.25 ± 0.42a 1.39 ± 0.56a Eosinophils (X 0.09 ± 0.04a 0.08 ± 0.02a 0.08 ± 0.00a 0.09 ± 0.03a 0.09 ± 0.05a 0.10 ± 0.04a 0.1 ± 0.05a 0.09 ± 0.04a 109/L) Basophils (X 109/L) 0.04 ± 0.02a 0.02 ± 0.01a 0.02 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a 0.02 ± 0.00a 0.03 ± 0.02a Haematocrit (L/L) 0.50 ± 0.01a 0.51 ± 0.03a 0.52 ± 0.01a 0.52 ± 0.02a 0.51 ± 0.00a 0.50 ± 0.00a 0.52 ± 0.02a 0.52 ± 0.00a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05). 191

Table 11. 5. Haematological values of rats in subacute toxicity of K. africana

Parameter Male Female Control 200 mg/Kg 400 mg/Kg 600 mg/Kg Control 200 mg/Kg 400 mg/Kg 600 mg/Kg WBC (X 109/L) 10.14 ± 10.37 ± 0.99a 10.25 ±0.85a 11.75 ± 7.42 ± 1.65a 9.9 ± 4.14a 7.02 ± 1.19a 8.29 ± 0.74a 2.35a 2.59ab RBC (X 1012/L) 7.74 ± 0.30a 8.43 ± 0.93a 8.09 ± 0.69a 8.61 ± 1.12a 8.15 ± 0.14a 8.28 ± 0.26a 8.72 ± 0.69a 8.37 ± 0.38a Haem (g/dL) 14.97 ± 15.23 ± 0.59a 15.1 ± 0.49a 16.4 ± 1.78a 14.97 ± 14.99 ± 15.09 ± 15.03 ± 0.31a 0.09a 0.06a 0.24a 0.09a MCV (fL) 64.93 ± 67.57 ± 3.59a 65.25 ± 66.8 ± 2.74a 63 ± 1.04a 67.83 ± 69.6 ± 9.67a 69.1 ± 9.63a 0.79a 1.28a 5.83a MCH (pg) 19.3 ± 0.36a 19.49 ± 0.48a 19.31 ± 19.37 ± 18.43 ± 18.63 ± 18.9 ± 0.79a 18.9 ± 0.82a 0.39a 0.42a 0.29a 0.46a MCHC (g/dL) 29.73 ± 30.9 ± 1.29a 30.32 ± 31.4 ± 1.95a 29.23 ± 32.97 ± 31.73 ± 31.57 ± 0.26a 0.85a 0.26a 3.97a 3.86bc 3.68c

RDW (%) 11.5 ± 0.14a 11.57 ± 0.21a 11.53 ± 11.63 ± 10.87 ± 10.57 ± 10.3 ± 0.14a 10.4 ± 0.35a 0.18a 0.28a 0.81a 0.29a Platelet Count (X 781 ± 65.31a 1033.33 ± 923.83 ± 764.67 ± 778.67 ± 807 ± 909.33 ± 750.67 ± 109/ L) 317.69a 208.14a 49.77a 92.32a 118.85a 224.68a 64.65a MPV (fL) 9.97 ± 0.05 a 9.99 ± 0.07a 9.92 ± 0.01a 9.97 ± 0.09a 9.27 ± 0.31a 8.98 ± 0.07a 8.93 ± 0.01a 8.96 ± 0.05a Neutrophils (X 0.41 ± 0.10a 0.38 ± 0.08a 0.30 ± 0.01a 0.42 ± 0.13a 0.64 ± 0.06a 0.79 ± 0.24a 0.74 ± 0.19a 0.62 ± 0.03a 109/L) Lymphocytes( X 6.32 ± 0.90a 6.63 ± 1.25a 6.48 ± 1.06a 5.82 ± 0.41a 4.84 ± 0.94a 4.90 ± 0.90a 6.85 ± 2.96a 7.00 ± 3.10a 109/L) Monocytes (X 2.09 ± 0.12a 1.98 ± 0.01a 1.98 ± 0.00a 2.05 ± 0.19a 1.34 ± 0.53a 1.46 ± 0.61a 1.6 ± 0.75a 1.53 ± 0.71a 109/L) Eosinophils (X 0.09 ± 0.04a 0.13 ± 0.07a 0.11 ± 0.06a 0.11 ± 0.05a 0.09 ± 0.05a 0.15 ± 0.10a 0.18 ± 0.12a 0.11 ± 0.05a 109/L) Basophils (X 109/L) 0.04 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a 0.23 ± 0.20a 0.02 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a 0.03 ± 0.00a Haematocrit (L/L) 0.50 ± 0.01a 0.49 ± 0.00a 0.50 ± 0.00a 0.52 ± 0.01a 0.51 ± 0.00a 0.50 ± 0.00a 0.5 ± 0.00a 0.50 ± 0.01a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

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Table 11. 6. Haematological values of rats in subacute toxicity of the combination of both plants

Parameter Male Female Control 200 mg/Kg 400 mg/Kg 600 mg/Kg Control 200 mg/Kg 400 mg/Kg 600 mg/Kg WBC (X 109/L) 10.14 ± 9.43 ± 10.68 ± 11.29 ± 7.42 ± 1.65a 10.39 ± 4.63a 7.02 ± 1.26b 9.59 ± 3.79a 0.74a 0.07aa 1.20a 1.88a RBC (X 1012/L) 7.74 ± 0.30a 8.8 ± 1.48a 8.88 ± 1.52a 8.83 ± 1.51a 8.15 ± 0.14a 8.48 ± 0.49a 8.84 ± 0.88a 8.69 ± 0.64a Haem (g/dL) 14.97 ± 16.57 ± 16.67 ± 16.77 ± 14.97 ± 15.87 ± 0.94a 15.77 ± 16.03 ± 0.31a 1.91a 1.99a 2.06a 0.09a 0.84a 1.13a MCV (fL) 64.93 ± 68.23 ± 69.33 ± 66.47 ± 63 ± 1.04a 67.87 ± 5.87a 65.37 ± 64.93 ± 0.79a 4.29b 4.34ab 2.25b 3.41a 2.93a MCH (pg) 19.3 ± 0.36a 19.27 ± 19.36 ± 19.37 ± 18.43 ± 18.7 ± 0.58b 18.8 ± 0.69a 18.43 ± 0.26a 0.37a 0.32a 0.29a 0.27a MCHC (g/dL) 29.73 ± 32.43 ± 31.63 ± 33.73 ± 29.23 ± 32.37 ± 3.48a 32.2 ± 3.36a 33.6 ± 3.91a 0.26a 2.94a 2.13a 4.29a 0.26a RDW (%) 11.5 ± 0.14a 13.37 ± 13.33 ± 15.5 ± 4.14a 10.87 ± 13.03 ± 3.01a 13.07 ± 16.73 ± 2.08a 2.03a 0.81a 3.09a 6.69a PC(X 109/ L) 781.3 ± 730.67 726 ± 10.07a 1034 ± 778.67 ± 898.67 ± 950.67 ± 1063 ± 65.31a ±15.01a 318.83a 92.32a 212.78b 264.28a 364.36a MPV (fL) 9.97 ± 0.05a 9.93 ± 0.01a 9.97 ± 0.02a 9.95 ± 0.01a 9.27 ± 0.31a 9.33 ± 0.39a 9.37 ± 0.44a 9.33± 0.41a Neutrophils (X 109/L) 0.41 ± 0.10a 0.62 ± 0.15a 0.99 ± 0.56a 1.27 ± 0.87a 0.64 ± 0.06a 0.63 ± 0.07a 0.73 ± 0.18a 1.22 ± 0.58a Lymphocytes( X 6.32 ± 0.90a 6.11 ± 0.64a 6.78 ± 1.40a 6.40 ± 0.99a 4.84 ± 0.94a 7.09 ± 3.14a 7.31 ± 3.38a 7.92 ± 1.97a 109/L) Monocytes (X 109/L) 2.09 ± 0.12a 2.01 ± 0.09a 2.06 ± 0.09a 2.07 ± 0.13a 1.34 ± 0.53a 1.39 ± 0.59a 1.32 ± 0.54a 1.39 ± 0.62a Eosinophils (X 109/L) 0.09 ± 0.04a 0.14 ± 0.07a 0.12 ± 0.05a 0.11 ± 0.05a 0.09 ± 0.05a 0.13 ± 0.08a 0.15 ± 0.09a 0.17 ± 0.11a Basophils (X 109/L) 0.04 ± 0.00a 0.06 ± 0.02a 0.05 ± 0.03a 0.06 ± 0.06a 0.02 ± 0.00a 0.03 ± 0.01a 0.03 ± 0.00a 0.04 ± 0.01a Haematocrit (L/L) 0.50 ± 0.01a 0.51 ± 0.01a 0.53 ± 0.02a 0.5 ± 0.03a 0.51 ± 0.00a 0.49 ± 0.00a 0.49 ± 0.02a 0.48 ± 0.02a Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

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Effect of V. mespilifolia, K. africana and the combination of both plants on blood chemistry parameters of rats after 28 days of treatment.

Tables 11.7, 11.8 and 11.9 showed the effect of daily administration of the aqueous extracts of

V. mespilifolia, K. africana and the combination of both plants for 28 days on body electrolyte levels, liver function enzymes and lipid profile on both sexes of rats. Transaminases (ALT and

AST) are good indices of liver damage (Arneson and Brickell, 2007). ALP is most often measured to indicate bile duct obstruction (Witthawaskul et al. 2003). In this study, there were no deleterious changes found in the levels of transaminases in the plasma of treated animals.

The results of the biochemical studies showed that there was no significant changes in the activities of serum ALT, GGT, AST and ALP at treatment doses of V. mespilifolia, K. africana and the combination of both plants on both sexes in comparison with their respective control groups (11.7, 11.8 and 11.9). The administration of V. mespilifolia, K. africana and the combination of both plants on both sexes of animals produced a non significant diference in the levels of serum total protein, albumin, total bilirubin, conjugated bilirubin, urea and creatinine in both female and males when compared with their respective control group. Renal damage is usually marked by increase in levels of creatinine (Gowda et al. 2010). The observed result in this study also suggests that the aqueous extract of these plants do not have a negative effect, but rather seems to have a renal protective. This is shown by its inhibitory effect on unnecessary degradation of protein that may be released due to cell damage caused by free radicals. Electrolyte functions are often considered when assessing kidney function. Increase or decrease in electrolytes such as sodium, potassium, chloride and magnesium ions could indicate renal injury (Oh, 2011). In this study, there was no significant difference in the serum level of sodium, chloride, glucose, calcium and magnesium in both female and male rats treated with V. mespilifolia, K. africana and the combination of both plants at all doses tested when

194

compared with their respective control groups (Tables 11.7, 11.8 and 11.9). Thus it suggest that the plants did not cause electrolyte imbalance.

The administration of the aqueous extracts of V. mespilifolia, K. africana and their combination on both sexes of rats produced no significant difference in the levels of triglyceride, high density level-cholesterol and c-reactive protein at all doses tested when compared with their respective control groups (Tables 11.7, 11.8 and 11.9). Finally, there was no significant difference in the serum level of total cholesterol in both female and male rats treated with V. mespilifolia and K. africana at all doses tested when compared with their respective control groups (Table 11.7 and 11.8) while the combination of both plants to both sexes of animals produced a significant decrease (p<0.05) in total cholesterol level in male rats and a non significant difference in female rats when compared to their respective control group (Table

11.9).

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Table 11. 7. Effect of daily administration of whole plants of V. mespilifolia extract for 28 days on biochemical profiles on both sexes of control and treated rats in sub-acute toxicity study.

Parameter Male Female Control 200 mg/kg 400 mg/kg 600 mg/kg Control 200 mg/kg 400 mg/kg 600 mg/kg Sodium (mmol/L) 138.8 ± 0.74 a 138.6 ± 0.70a 139.2 ± 1.14 a 139.8 ± 1.18a 140.2 ± 1.72a 139.4 ± 0.95a 139.8 ± 1.39a 139.7 ± 1.22a Chloride (mmol/L) 103 ± 1.10a 102 ± 0.99a 101.9 ± 0.01a 104.4 ± 2.49a 107.4 ± 3.2a 105.2 ± 1.03a 105.6 ±1.38a 106.8 ± 2.57a Urea (mmol/L) 4.58 ± 0.28a 4.94 ± 0.68a 4.44 ± 0.15a 4.8 ± 0.55a 5.38 ± 0.66a 6.86 ± 2.19a 6.02 ± 1.32a 6.22 ± 1.46a Creatinine (umol/L) 30.2 ± 3.37a 28.2 ± 2.03a 27.6 ± 0.71a 30.8 ± 3.93a 37.8 ± 5.04a 34.8 ± 2.04a 35.8 ± 3.07a 35.2 ± 2.99a Glucose 5.3 ± 0.38a 4.94 ± 0.03a 5.04 ± 0.17ab 5.35 ± 0.40a 5.6 ± 0.58a 5.74 ± 0.70a 5.72 ± 0.70a 5.74 ± 0.75a Calcium (mmol/L) 2.47 ± 0.03a 2.35 ± 0.10b 2.46 ± 0.09a 2.46 ± 0.02a 2.27 ± 0.21a 2.32 ± 0.26a 2.08 ± 0.03a 2.38 ± 0.29a Magnesium 0.97 ± 0.06a 1.02 ± 0.13a 0.97 ± 0.03a 0.99 ± 0.05a 1.09 ± 0.05a 1.02 ± 0.03a 1.05 ± 0.09a 1.08 ± 0.07a (mmol/L) Uric acid (mmol/L) 0.13 ± 0.02a 0.19 ± 0.07a 0.13 ± 0.01a 0.12 ± 0.01a 0.28 ± 0.05a 0.2 ± 0.00a 0.23 ± 0.01a 0.27 ± 0.03a Total Protein (g/L) 52.2 ± 1.83a 51.4 ± 1.05a 51.6 ± 1.03a 51.4 ± 1.08a 48.2 ± 2.14a 48.6 ± 2.06a 51 ± 2.83a 48.6 ± 1.74a Albumin (g/L) 17.8± 0.4a 18.2 ± 0.8a 18.6 ± 0.89a 18.6 ± 0.91a 20 ± 0.63a 19.4 ± 0.8a 20 ± 0.89a 19.2 ± 0.98a Total bil (umol/L) 14.8 ± 3.71a 17.2 ± 6.16a 14.6 ± 3.68a 14.6 ± 3.61a 24 ± 6.36a 21 ± 3.39a 23 ± 5.36a 24.4 ± 6.39a Con bil (umol/L) 5.4 ± 2.15a 7.2 ± 3.97a 4.8 ± 1.55a 5.6 ± 2.31a 8.6 ± 1.62a 8.4 ± 1.50a 9.4 ± 2.45a 8.9 ± 1.92a ALT (U/L) 52.8 ±5.04 a 55.76 ± 7.96a 55.6 ± 7.59a 62 ± 14.24a 74.2 ± 11.51a 64.2 ± 2.91a 76.2 ± 13.89a 79.4 ± 16.44a AST (U/L) 159.6 ± 20.5a 149.2 ± 10.4a 161.8 ± 148.2 ± 226.8 ± 246.4 ± 226 ± 42.35a 251.2 ± 68.69a 22.73a 10.32a 43.07a 62.64a ALP (U/L) 253.4 ± 268 ± 28.04a 256.8 ± 259.6 ± 152.8 ± 151.4 ± 147.2 ± 142 ± 15.21a 13.46a 15.54a 18.70a 25.21a 24.56a 20.33a Total chol (mmol/L) 1.06 ± 0.03a 1.09 ± 0.12a 1.06 ± 0.09a 1.02 ± 0.09a 0.93 ± 0.11a 1.13 ± 0.36a 1.03 ± 0.22a 0.98 ± 0.15a TAG (mmol/L) 2.14 ± 0.42a 1.72 ± 0.02a 1.93 ± 0.26a 1.95 ± 0.25a 1.56 ± 0.17a 1.59 ± 0.19a 1.56 ± 0.15a 1.54 ± 0.16a HDL chol (mmol/L) 0.75 ± 0.03a 0.81 ± 0.08a 0.81± 0.07a 0.83 ± 0.09a 0.80 ± 0.04a 0.84 ± 0.07a 0.85 ± 0.09a 0.78 ± 0.01a CRP (U/L) < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 GGT (U/L) < 5 < 5 < 5 < 5 < 5 < 5 < 5 < 5 Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

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Table 11. 8. Effect of daily administration of tubers of K.africana extract for 28 days on biochemical profiles on both sexes of control and treated rats in sub-acute toxicity study.

Parameter Male Female Control 200 mg/kg 400 mg/kg 600 mg/kg Control 200 mg/kg 400 mg/kg 600 mg/kg Sodium (mmol/L) 138.8 ± 0.74a 138.2 ± 0.16a 138.8 ± 0.74a 139 ± 0.79a 140.2 ± 1.72a 139.4 ± 0.92a 138.9 ± 0.43a 138.6 ± 0.12a Chloride (mmol/L) 103 ± 1.10a 103.8 ± 1.93a 104.8 ± 2.87a 103 ± 1.09a 107.4 ± 3.2a 105.2 ± 0.98a 104.6 ± 0.40a 105 ± 0.79a Urea (mmol/L) 4.58 ± 0.28a 5.48 ± 1.18a 6.14 ± 1.84b 5.56 ± 1.32a 5.38 ± 0.66a 5.98 ± 1.23a 6.4 ± 1.64a 6.96 ± 2.22a Creatinine (umol/L) 30.2 ± 3.37a 27 ± 0.16a 27.8 ± 0.98a 27.4 ± 0.59a 37.8 ± 5.04a 33.6 ± 0.82a 35 ± 2.24a 38 ± 5.23a Glucose 5.3 ± 0.38a 5.02 ± 0.08a 4.9 ± 0.17a 4.86 ± 0.16a 5.6 ± 0.58a 5.33 ± 0.35a 5.78 ± 0.73a 5.65 ± 0.62a Calcium (mmol/L) 2.47 ± 0.03a 2.47 ± 0.03a 2.48 ± 0.05a 2.54 ± 0.09a 2.27 ± 0.21a 2.40 ± 0.35a 2.49 ± 0.45a 2.47 ± 0.42a Magnesium (mmol/L) 0.97 ± 0.06a 1.08 ± 0.19a 1.02 ± 0.08a 1.06 ± 0.16a 1.09 ± 0.05a 1.02 ± 0.00a 1.11 ± 0.08a 1.04 ± 0.01a Uric acid (mmol/L) 0.13 ± 0.02a 0.19 ± 0.07a 0.17 ± 0.05a 0.16 ± 0.03a 0.28 ± 0.05a 0.27 ± 0.03a 0.29 ± 0.06a 0.28± 0.05a Total Protein (g/L) 52.2 ± 1.83a 52 ± 1.63a 52 ± 1.61a 51.6 ± 1.25a 48.2 ± 2.14a 50 ± 3.96a 51.2 ± 5.14a 50.8 ± 4.74a Albumin (g/L) 17.8± 0.4a 18.2 ± 0.75a 18 ± 0.63a 17.6 ± 0.20a 20 ± 0.93a 19.4 ± 0.05a 19.8 ± 0.41a 19.52 ± 0.14a Total bil (umol/L) 14.8 ± 3.71a 17.2 ± 6.09a 12.4 ± 1.28a 12.8 ± 1.68a 24 ± 6.36a 19 ± 1.38a 22.2 ± 4.58a 26.4 ± 8.76a Con bil (umol/L) 5.4 ± 1.07a 5.60 ± 1.25a 5.82 ± 1.49a 5.6 ± 1.28a 8.6 ± 1.62a 7 ± 0.03a 7.98 ± 1.00a 8.49 ± 1.51a ALT (U/L) 52.8 ±5.04a 56.2 ± 8.44a 58 ± 10.19a 62.4 ± 12.06a 74.2 ± 11.51a 70.6 ± 7.92a 74.6 ± 11.89a 76.8 ± 14.10a AST (U/L) 159.6 ± 20.48a 178.6 ± 36.43a 177.6 ± 35.52a 176 ± 34.84a 226.8 ± 222.8 ± 290.4 ± 271.4 ± 43.07a 39.07a 106.65a 87.65a ALP (U/L) 253.4 ± 13.46a 245.8 ± 5.79a 251.6 ± 11.67a 257.6 ±17.62a 152.8 ±25.21a 140.6 ±12.98a 152 ± 24.39a 149.6 ± 22.03a Total chol (mmol/L) 1.06 ± 0.03a 1.18 ± 0.15a 1.12 ± 0.12a 1.2 ± 0.16a 0.93 ± 0.11a 1.09 ± 0.24a 1.06 ± 0.24a 1.04 ± 0.22a TAG (mmol/L) 2.14 ± 0.42a 1.76 ± 0.02a 1.81 ± 0.07a 2.15 ± 0.42a 1.56 ± 0.17a 1.58 ± 0.16a 1.56 ± 0.18a 1.59± 0.18a HDL chol (mmol/L) 0.75 ± 0.03a 0.81 ± 0.07a 0.82 ± 0.09a 0.80 ± 0.07a 0.80 ± 0.04a 0.79 ± 0.08a 0.82 ± 0.04a 0.81 ± 0.09a CRP (U/L) < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 GGT (U/L) < 5 < 5 < 5 < 5 < 5 < 5 < 5 < 5 Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05). 197

Table 11. 9. Effect of daily administration of the combination of both plants extract for 28 days on biochemical profiles on both sexes of control and treated rats in sub-acute toxicity study.

Parameter Male Female Control 200 mg/kg 400 mg/kg 600 mg/kg Control 200 mg/kg 400 mg/kg 600 mg/kg Sodium (mmol/L) 138.8 ± 0.74a 140.6 ± 2.52a 140 ± 1.94a 141 ± 2.92a 140.2 ± 1.72ab 138.6 ± 0.12a 140 ± 1.53a 140.4 ± 1.90a Chloride (mmol/L) 103 ± 1.10a 103.2 ± 1.25a 103.8 ± 1.33a 103.2 ± 1.88a 107.4 ± 3.2a 104.6 ± 0.08a 105.4 ± 1.19a 104.2 ± 0.01a Urea (mmol/L) 4.58 ± 0.28a 5.3 ± 0.98a 6.34 ± 2.04a 5.68 ± 1.42a 5.38 ± 0.66a 5.92 ± 1.20a 6.12 ± 1.39a 6.34 ± 1.60a Creatinine (umol/L) 30.2 ± 3.37a 31.6 ± 4.76a 30.2 ± 3.36a 32.4 ± 5.54a 37.8 ± 5.04a 33.2 ± 0.44a 33.4 ± 0.62a 36 ± 3.23a Glucose 5.3 ± 0.38a 4.92 ± 0.00a 5.04 ± 0.13a 5.3 ± 0.36a 5.6 ± 0.58a 5.7 ± 0.68a 5.76 ± 0.72a 5.56 ± 0.54a Calcium (mmol/L) 2.47 ± 0.03a 2.45 ± 0.01a 2.51 ± 0.07a 2.48 ± 0.06a 2.27 ± 0.21a 2.39 ± 0.34a 2.38 ± 0.36a 2.44 ± 0.40a Magnesium (mmol/L) 0.97± 0.06a 1.18 ± 0.29a 1.05 ± 0.12a 1.10 ± 0.17a 1.09 ± 0.05a 1.04 ± 0.00a 1.06 ± 0.01a 1.06 ± 0.02a Uric acid (mmol/L) 0.13 ± 0.02a 0.21 ± 0.11a 0.18 ± 0.06a 0.20 ± 0.08a 0.28 ± 0.05a 0.26 ± 0.02a 0.29 ± 0.06a 0.30 ± 0.07a Total Protein (g/L) 52.2 ± 1.83a 54.2 ± 3.79a 54.2 ± 3.82a 55 ± 4.59a 48.2 ± 2.14a 52.6 ± 6.52a 53.6 ± 7.52a 54.4 ± 4.18a Albumin (g/L) 17.8± 0.4a 18.6 ± 1.19a 18.6 ± 1.17a 19 ± 1.60a 20 ± 0.63a 19.6 ± 0.21a 20.4 ± 1.01a 20.6 ± 1.26a Total bil (umol/L) 14.8 ± 3.71a 14.2 ± 3.09a 14.6 ± 3.49a 14.6 ± 3.51a 24 ± 8.36a 25 ± 7.34a 26.6 ± 8.98a 26.4 ± 8.80a Con bil (umol/L) 5.4 ± 2.15a 6.8 ± 3.53a 6.8 ± 3.51a 6.9 ± 3.63a 8.6 ± 1.62a 7.2 ± 0.22a 7.9 ± 0.90a 8.8 ± 1.83a ALT (U/L) 52.8 ±5.04a 61 ± 13.26a 66.2 ± 18.39a 73.4 ± 25.61a 74.2 ± 1.51a 70.6 ± 7.91a 76.8 ± 14.09a 79.8 ± 17.08a AST (U/L) 159.6 ± 20.48a 173.8 ± 34.68a 184.2 ± 45.06a 225 ± 85.79a 226.8 ± 43.07a 199 ± 15.29a 216.6 ± 32.84a 226 ± 42.28a ALP (U/L) 253.4 ± 13.46a 246.4 ± 6.45a 249 ± 9.06a 256.6 ± 16.69a 152.8 ± 25.21a 128.6 ± 1.01a 136.2 ± 8.59a 136.4 ± 8.83a TC (mmol/L) 1.06 ± 0.03a 1.12 ± 0.19b 1.02 ± 0.08b 0.99 ± 0.08b 0.93 ± 0.11a 0.99 ± 0.17a 0.95 ± 0.16a 0.99 ± 0.17a TAG (mmol/L) 2.14 ± 0.42a 1.76 ± 0.69a 1.60 ± 0.54a 2.17 ± 1.12a 1.66 ± 0.57a 1.62 ± 0.53a 1.70 ± 0.59a 1.75 ± 0.67a HDL chol (mmol/L) 0.75 ± 0.03a 0.79 ± 0.05a 0.75 ± 0.06a 0.69 ± 0.00a 0.80 ± 0.04a 0.76 ± 0.00a 0.81 ± 0.06a 0.82 ± 0.06a CRP (U/L) < 1 < 1 < 1 < 1 < 1 < 1 < 1 < 1 GGT (U/L) < 5 < 5 < 5 < 5 < 5 < 5 < 5 < 5 Data are presented as mean ± SD (n = 5). Values with the superscripts in the same row for each parameter are not significantly different (p > 0.05).

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Results of the histopathological studies of the tissue sections of the liver and kidney of male and female rats administered with 600 mg/kg/bwt aqueous extracts of V. mespilifolia and K. africana respectively, and the combination of both plants showed well differentiated hepatocyte. Furthermore, renal epithelial cells were well developed and show no degenerative or inflammatory lesions (Figure 11.1and 11.2).

Figure 11. 1: Photomicrographs of liver and kidney of male rats displaying the control (a and b), groups treated with 600 mg/kg of V. mespilifolia (c and d), K. africana (e and f) and the combination of both plants (g and h) section respectively (H&E X400). 199

Figure 11. 2: Photomicrographs of liver and kidney of female rats displaying the control (a and b), groups treated with 600 mg/kg of V.mespilifolia (c and d), K. africana (e and f) and the combination of both plants (g and h) section respectively (H&E X400). 200

CONCLUSION The results of the in vivo acute and subchronic toxicity evaluation suggested that the aqueous extracts of the V.mespilifolia, K. africana and the combination of both plants up to the dose of

600 mg/kgb.wt is relatively safe when administered orally to female and male rats. This can be deduced from the fact that the extract at the different doses did not show any lethality or adverse effects on the rats, and did not induce significant alterations in all the biochemical, haematological and morphological parameters investigated in this study, regardless of gender.

This could be an assurance of the oral safety of V.mespilifolia, K. africana and the combination of both plants at the doses tested as practiced traditionally in folk medicine.

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CHAPTER TWELVE

Evaluation of the anti-obesity potential of Kedrostis africana, Vernonia mespilifolia and the combination of both plants using enzyme-based in-vitro assays

205

Chapter Twelve

Evaluation of the anti-obesity potential of Kedrostis africana, Vernonia mespilifolia and the combination of both plants using enzyme-based in-vitro assays

Contents Pages

Introduction ...... 207

Materials and methods ...... 208

Results and discussion ...... 212

Conclusion ...... 218

References ...... 219

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INTRODUCTION Obesity is a complex metabolic disorder which has turned out to be a worldwide epidemic, because of its implication in the onset of several diseases, such as type 2 diabetes, hypertension, coronary heart diseases, metabolic syndrome, stroke, and some cancers (Barnes, 2015).

Therefore, prevention and treatment of obesity has become one of greatest public health challenges of the 21st century. The reduction of nutrient digestion and absorption by developing of enzyme inhibitors without altering major mechanism in gastrointestinal system has become one of the most important strategies in the treatment of obesity (Deshpande et al. 2013). In recent times, a number of studies on the prevention and treatment of obesity are on the rise

(Deshpande et al. 2013). Some of these include suppression on food intake, stimulation of energy expenditure, lipase inhibition, regulation of lipid metabolism, and inhibition of adipocyte differentiation (Yun, 2010). Pancreatic lipase and α-amylase are typical enzymes that can be inhibited. These enzymes are important in the lipopytic and glycosidic chain hydrolysis for efficient digestion of triglycerides, starch and glycogen (Yun, 2010). Therefore, compounds that could inhibit pancreatic lipase and α- amylase activities would be useful in the treatment of obesity thus resulting in the reduction of calories and intake of food (Zhang and

Lu, 2012). One of the commercially available anti-obesity drug, Orlistat acts by inhibiting pancreatic lipase activity but it has adverse side effects such as abdominal pain, bloating, flatulence, oily stools, diarrhea, and decreasing in fat soluble vitamins absorption (Kang and

Park, 2012). Acarbose on the other hand is a reversible inhibitor of α-amylase and α- glucosidase with also undesirable side effects limit its use (Supkamonseni et al. 2014). Many plants and their active chemical compounds have demonstrated activity in the treatment of obesity that have fewer side effect and less toxicity compare to synthetic drugs (Gulati et al.

2012; Sivasangari et al. 2014). This study investigated the potential of K. africana, V.

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mespilifolia and their combination to inhibit these enzymes as an indication of their anti-obesity effects.

MATERIALS AND METHODS Collection and extraction of sample were carried out as previously described in chapters

5 but only the aqueous and ethanol extracts were used in this chapter because they are the solvents used according to the ethnobotanical survey carried out.

Inhibition of Lipase activity

Principle Lipase plays a key role in fat digestion by hydrolyzing dietary triglycerides to monoglycerides and free fatty acids (Abel et al. 2001). Inhibition of lipase activity has been explored as a possible mechanism for reducing total caloric intake and therefore, a possible means of interfering with the development of obesity (Lewis and Liu, 2012). In this assay, the conversion of the substrate, para-Nitrophenol palmitate (pNPP) was used to study lipase inhibition in a single phase system.

Reagents i. Solution A (pNPP): 2 mg pNPP was dissolved in 2 mL of 10% isopropanol. ii. Solution B: consisted of 20 mg gum arabic, 40 mg sodium deoxycholate and 100 μl of Triton

X-100 all dissolved in 18 ml of Tris-HCl buffer (pH 8.0). iii. Substrate solution: Solution A was added to solution B and stirred until all was dissolved. iv. Lipase solution (10 mg/mL): 50 mg of porcine pancreatic lipase added to 5 ml of 50 mM

Tris-HCl buffer.

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Procedure The lipase inhibition assay was conducted according to the method described by Zhang et al.

(2011) with some modifications. Ten microlitre of extracts (prepared at concentrations of 50,

100 and 200 μg/mL), positive control (Orlistat, 100 μM) and DMSO (negative control vehicle used to dissolve the extracts) were pipetted into respective wells of a 96-well plate. Freshly prepared porcine pancreatic lipase was added at four times the volume of the test samples, positive and negative controls (40 μL). The plates were initially incubated at 37ºC for 15 minutes. Thereafter, 170 μL of the substrate solution was added to the wells. The plate was then incubated at 37°C for 25 minutes and the absorbance was read at 405 nm using a microplate reader (Diagnostic Automation, Inc, USA, DAR 800).

Percentage lipase inhibition was calculated as:

% Lipase inhibition = 1- (A/B) X 100.

Where A = absorbance of test well (plant extract or orlistat)

B = Average absorbance of enzyme control

Alpha amylase inhibition assay Assay Principle: Alpha amylase activity was measured by the amount of starch hydrolyzed into monosaccharides in the presence of the enzyme. The reaction incorporates an iodine reagent which gives a blue color in the presence of starch. In the presence of an enzyme inhibitor, the intensity of the color, measured spectrophotometrically, indicates the amount of starch remaining in the reaction mixture, and hence, the extent of alpha amylase inhibition.

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Reagents i. Starch solution (2 mg/mL): 0.2 g of starch in 100 mL distilled water. Solubilization was achieved by boiling the starch solution in a glass beaker directly on a stirring plate for 15 minutes.

ii. Phosphate buffer: 1.2 g NaH2PO4 and 0.2 g NaCl was dissolved in 450 mL distilled water. pH was adjusted to 6.0 using 1M NaOH, and volume was made up to 500 mL. iii. 1M Hydrochloric acid (stock solution=37%): 8.2 mL HCl (1N) was added to 25 mL distilled water, made up to 100 mL with same.

iv. Iodine reagent: 0.127 g I2 and 0.083 KI were mixed together in 100 ml of distilled water. v. Alpha amylase enzyme: 10 mg porcine pancreatic amylase was solubilized in 100 mL phosphate buffer. The preparation was made just before use and kept on ice. vi. Acarbose stock solution (positive control): A 500 μM solution was initially prepared by mixing 3.2 mg acarbose dissolved in 10 ml phosphate buffer and aliquots of 1 mL were made into eppendorf tubes. This was stored at -20ºC until use. The aliquots were diluted five-fold to give a working concentration of 100 mM which is 64 µg/mL.

Procedure α-Amylase inhibitory activity was determined using the method of Zhang et al. (2011) with a slight modification. Plant extracts were prepared at the concentrations: 50, 100 and 200 μg/mL in a phosphate buffer. Ten microlitre of enzyme solution was pipetted into appropriate wells of a 96-well plate. Thereafter, 15 μL of test samples, phosphate buffer (the blank, enzyme without inhibitor) or positive control (acarbose, 64 µg/mL) were added to the enzyme in respective wells. The plate was pre-incubated for 10 minutes at 37ºC to allow interaction of the enzyme with the different compounds. The reaction was started by the addition of 40 μL starch solution

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to the wells, and the plate was again incubated for 30 minutes at 37ºC. The reaction was terminated by adding 20 μL of 1M HCl to each well, followed by 75 μL iodine reagent.

Absorbance was measured at 580 nm using a microplate reader (Diagnostic Automation, Inc,

USA, DAR 800). Alpha amylase inhibition was measured as a percentage of the enzyme control using the formula:

% α-amylase inhibition = 1- (B/A) X 100

Where A = absorbw2ance of test well (plant extract or acarbose)

B = Average absorbance of enzyme control

Controls without enzyme (no-enzyme) and without starch (no-starch) were also included in the assay to be certain that no reaction occurred when one of either the enzyme or substrate was absent. This was done to exclude false positive results, as some plants extracts have been reported to contain traces of α-amylase or starch.

Alpha glucosidase inhibition assay Assay Principle: The assay is based on the hydrolysis of p-nitrophenyl-α-D-glucopyranoside

(PNP-GLUC) specifically by α-glucosidase into a yellow colored product, p-nitrophenol (PNP) and D-glucose, with absorbance maximum at 405 nm. Inhibition of α-glucosidase results in reduced formation of PNP.

Reagents i. Potassium phosphate buffer (67 mM; pH 6.8) ii. p-Nitrophenyl-α-D-glucopyranoside (PNP-GLUC) solution (10 mM) iii. Sodium carbonate solution (100 mM) iv. α-glucosidase solution 50 µg/mL

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v. Epigallocatechin gallate; EGCG, 10 μg/mL (positive control)

Procedure α-Glucosidase inhibitory activity was determined according to a previously reported method with slight modifications (Zhang et al., 2011). Ten microlitre of plant extract (prepared at concentrations of 50, 100 and 200 μg/mL) or the positive control (EGCG) was mixed with 40

μL of α-glucosidase solution. This was pre-incubated at 37ºC for 5 minutes and initial background absorbance was read at 405 nm. 10 μL of PNP-GLUC was then added and the reaction mixture was incubated again for 20 minutes at 37ºC. The reaction was terminated by the addition of 50 μL of sodium carbonate solution. The absorbance was measured again at 405 nm using a microplate reader (Diagnostic Automation, Inc, USA, DAR 800). Controls without enzyme and without the substrate (PNP-GLUC) were also included in the assay.

The percentage inhibition of α-glucosidase was calculated as follows:

% α-glucosidase inhibition = 1- (B/A) X 100.

Where A = absorbance of test well (plant extract or positive control)

B = Average absorbance of enzyme control

Statistical analysis

All data in triplicates were subjected to one-way analysis of variance using MINITAB version

17 and the means separated by Fisher’s least significant differences (LSD) test (p ≤ 0.05).

RESULTS AND DISCUSSION Inhibition of pancreatic lipase activity Pancreatic lipase play a vital role in the hydrolysis of 50% to 70% of total dietary fats and triacylglycerols (Birari and Bhutani, 2007). It is mainly involved in the absorption and 212

digestion of triglycerides (TG) into monoglycerides and fatty acids (Kim et al. 2011). Research has shown that very few plant extracts exhibit very potent lipase activities (Seyedan et al. 2015) in comparison with established inhibitors such as Orlistat. Figure 1 a, b and c summarizes the results of pancreatic lipase inhibitory activities of the aqueous and ethanol extracts of V. mespilifolia, K. africana and the combination of both plants with orlistat as positive control.

The result obtained in this study revealed that both extracts of V. mespilifolia, K. africana and the combination of both plants showed weak inhibitory activities against porcine pancreatic lipase as shown in Figure 7.1. Pancreatic lipase inhibition by the extracts was in the order ethanol extracts of the combination of both plants > ethanol of K. africana > ethanol of V. mespilifolia > aqueous extract of the combination of both plants > aqueous extract of K. africana > aqueous V. mespilifolia. The highest inhibition was obtained with the ethanol extract of combination of both plants (21.19%). This was much lower than the value obtained for

Orlistat (72.98%) the positive control. The ethanol extracts of all the tested samples exhibited greater inhibitory potential when compared with aqueous extracts. Furthermore, enzyme stimulation was observed at 50 µg/mL in ethanol extracts of V. mespilifolia and K. africana, as well as the aqueous extract of the combination of both plant (Figure 12.1a-c). Several studies have reported the inhibitory effects of medicinal plant extracts against pancreatic lipase (Kim and Kang, 2005; Dechakhampu and Wongchum, 2015). We report for the first time to the best of our knowledge, the pancreatic lipase inhibitory activities of the aqueous and ethanol extracts of V. mespilifolia, K. africana and their combination. It can be deduced from this study that all the extract tested have moderate inhibitory activities against porcine pancreatic lipase as shown in Figure 12.1.

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80 @ 70 60 @ 50 @ 40 @ 30 a a 20 a b c 10 b 0 b -10 Control Orlistat 100 50 μg/mL 100 μg/mL 200 μg/mL Lipase Inhibition (%) Inhibition Lipase -20 μM -30 Control Orlistat V. mespilifolia K. africana Combination

80 @ 70 60 50 @ 40 @ @ 30 a b c 20 c c b 10 a a b 0

-10 Control Orlistat 100 50 μg/mL 100 μg/mL 200 μg/mL Lipase Inhibition (%) Inhibition Lipase -20 μM -30 Control Orlistat V. mespilifolia K. africana Combination

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Inhibition of alpha-amylase In recent times, different studies have revealed the correlation between excessive intake of calories and myraids of chronic diseases, such as obesity, type 2 diabetes mellitus (T2DM), hyperlipidemia, and cardiovascular diseases (Etoundi et al. 2010; Bajwa et al. 2013; Upadhyay,

2015). Obesity happens to be the most common complication of hyperlipidemia and T2DM, since they share similar causative factors, chemical abnormalities, and clinical complications

(Shamseddeen et al. 2011). α- glucosidase and α-amylase are the key enzymes in carbohydrate digestion. The inhibition of these two enzymes can delay carbohydrate digestion thus reducing the rate of glucose absorption in the body (Kim et al. 2011b). The ability of the crude extracts of V. mespilifolia, K. africana, and combination of both plants to inhibit α-amylase is shown in Figure 12.2. All the extracts exhibited α-amylase inhibition in a dose dependent manner. The aqueous extract of K. africana exhibited the highest inhibition (31.64%) of α-amylase which was followed by both aqueous (25.28%) and ethanol (25.77%) extracts of the combination of both plants while the percentage inhibition of the positive control (acarbose) was 92.79% //at a concentration of 64 µg/mL. Inhibiting carbohydrate-hydrolyzing enzymes such as the amylases reduces the absorption of glucose because it help in reducing insulin sensitivity which is an hallmark of diet-induced obesity (Liu et al. 2011; Hamden et al. 2011). It is also vital in the reduction of precusors necessary for the synthesis and accumulation of triacylglycerol by the adipose tissue, thus, amylases could serve as a veritable target for obesity treatment

(Egedigwe et al. 2016). Barrett and Udani (2011) have revealed the crucial role plant extracts play in the inhibition of the absorption of glucose from the intestines and modulation of disorders related to energy metabolism.

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100 #

60 # # 40 # b b c b c a 20 a a a

amylase inhibition (%) inhibition amylase -

α Control Acarbose 64 50 μg/mL 100 μg/mL 200 μg/mL -20 µg/mL Control Acarbose V. mespilifolia K. africana Combination b)

100 #

60 # # 40 # a a c a b 20 a b c b

amylase inhibition (%) inhibition amylase 0 -

α Control Acarbose 64 50 μg/mL 100 μg/mL 200 μg/mL -20 µg/mL Control Acarbose V. mespilifolia K. africana Combination

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Inhibition of alpha-glucosidase Figure 12.3 summarizes the results of α- glucosidase inhibitory activities of the aqueous and ethanol extracts of V. mespilifolia, K. africana, and combination of both plants with

Epigallocatechin gallate (EGCG) as positive controls. A dose dependent inhibition was also observed in α- glucosidase activity for all the tested extracts. A stronger inhibition was observed when compared with α- amylase inhibition. Thus it could suggested α-amylase inhibition may not be the mechanism of action of the extracts. The ethanol extract of K. africana had the highest inhibitory potential (54.30%), followed by the ethanol extracts of the combination of both plants (47.36%) and the aqueous extract of K. africana while the percentage inhibition of the positive control (EGCG) was 67.57% at a concentration of 10

µg/mL. The findings from this study is in line with earlier reports by Kwon et al. (2007) that most natural α-glucosidase inhibitors from plants exhibited strong inhibitory activity against α- glucosidase and could be exploited as therapeutic agents for the management of postprandial hyperglycemia with slight side effects. a)

80 70 $ 60 50 $ $ 40 $ b c b a c a 30 b b 20 a 10

0 glucosidase inhibition(%) glucosidase - -10 Control EGCG 10 50 μg/mL 100 μg/mL 200 μg/mL α μg/mL

Control EGCG V. mespilifolia K. africana Combination

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80 $ 70 $ $ 60 b $ b 50 c c 40 b a a (%) 30 a c 20

glucosidase inhibition glucosidase -

α 0 -10 Control EGCG 10 50 μg/mL 100 μg/mL 200 μg/mL μg/mL Control EGCG V. mespilifolia K. africana Combination

Figure 12. 3: Inhibition of alpha glucosidase activity of a) aqueous extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. b) ethanol extracts of V. mespilifolia, K. africana, and combination of both plants at different concentrations. Values are mean ± SD (n = 3). Mean separation by LSD (p < 0.05). Set of bars (the same concentration) with different alphabets are different. $Lower than EGCG (positive control).

CONCLUSION This study is the first in- vitro anti-obesity report on V. mespilifolia, K. africana, and combination of both plants. The aqueous extract of K. africana exhibited the best α- amylase inhibitory activity while the ethanol extracts of V. mespilifolia and combination of both plants all the tested plants exhibited the best α- glucosidase and lipase inhibitory activities respectively. This supports earlier reports in this study where the ethanol extract exhibited the highest anti-oxidant activity (Chapter 4). However, this inhibitory effect is moderate when compared to the standards used. This study tend to suggests that the mechanisms by which the two plants under study exert their anti-obesity effect is probably not through the inhibition of key enzymes of metabolism such as pancreatic lipase, α- amylase and α- glucosidase inhibition.

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Kahn, B.B., 2001. Adipose-selective targeting of the GLUT 4 gene impairs insulin

action in muscle and liver. Nature 409, 729–733.

Bajwa, S.J., Sethia, E., Kaur, R., 2013. Nutritional risk factors in endocrine diseases. Journal

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potential. Drug Discovery Today 12, 879–889.

Dechakhamphu, A., Wongchum, N., 2015. Screening for anti-pancreatic lipase properties of

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Egedigwe, C.A., Ijeh, I.I., P.N., Okafor; Ejike C.E.C.C., 2016. Aqueous and methanol extracts

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modulation of appetite-regulatory hormones. Pharmaceutical Biology, 54(12),

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Etoundi, C.B., Kuaté, D., Ngondi, J.L., Oben, J., 2010.Anti-amylase, anti-lipase and

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Gulati, V., Harding, I., Palombo E., 2012. Enzyme inhibitory and antioxidant activities of

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Hamden K, Keskes H, Belhaj S, Mnafgui, K., Feki, A., Allouche, N., 2011. Inhibitory potential

of omega-3 fatty and fenugreek essential oil on key enzymes of carbohydrate-

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sorghum, foxtail millet and proso milleton α-glucosidase and α-amylase activities.

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Kwon, O., Eck, P., Chen, S., Corpe, C.P., Lee, J.H., Kruhlak, M., Levine, M., 2007. Inhibition

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Lewis, D.R., Liu, D.J., 2012. Direct measurement of lipase inhibition by Orlistat using a

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CHAPTER THIRTEEN

General Discussion and Conclusion

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GENERAL DISCUSSION AND CONCLUSION

DISCUSSION The main goal of anti-obesity therapy is to reduce or control body weight gain of obese or overweight subjects to normal weight levels. Conventional anti-obesity agents act via numerous mechanisms to achieve their therapeutic goals. These mechanisms may include the reduction in lipid absorption, decrease in energy intake, increase energy expenditure, decrease in pre-adipocyte differentiation and proliferation, decrease in lipogenesis and increase lipolysis, reduction of oxidative stress and the inhibition of processes involved in development of obesity complications (Mohamed et al. 2014; Sun et al. 2016). Majority of the available medications, however pose a lot of unpleasant side-effects and are usually very expensive.

Hence, the is need for the development of a safe, cost-efficient and pharmacologically-effective alternative medicines for the treatment or management of obesity.

Many communities in Africa are seeking alternatives to conventional anti-obesity therapy from natural sources, especially plants. In the Eastern Cape Province of South Africa, traditional healers have utilized indigenous plants for the management or treatment of obesity, and a number of these plants have been documented in a number of ethnobotanical surveys (Afolayan and Mbaebie, 2010; George and Nimmi, 2011).

Kedrostis africana, Vernonia mespilifolia and the combination of the two plants in equal ratio are among the most frequently mentioned plants in this province. These plants were chosen for this research as there are no available scientific reports on their chemical composition, toxicology or the nature of their acclaimed anti-obesity actions. Thus this research is centred on providing information that could either validate or nullify the ethno-medicinal claims for the use of these plants in the management or treatment of obesity. The whole plant extracts of

V. mespilifolia and the tuber of K. africana were employed as they are the parts mostly used 224

traditionally. Despite their renowned medicinal potentials in the Eastern Cape, little or no report is available in the literature concerning nutritional content and biological activities. Hence this study analyzed the proximate parameters, anti-nutritional content and mineral compositions of both plants. Both plants had abundant amount of important mineral ( e.g. Ca, Mg, Fe, P, Na,

Cu and Mn) and proximal contents (e.g. crude protein, crude fibre and crude fat) required daily by the human body. The presence of these components could be the underlining factors for their wide therapeutic applications in ethnomedicine. Quantitative analysis of crude extracts from both plants and their combination revealed the presence of essential polyphenolic like flavonoids, phenols, proanthocyanidins and tannins which have been shown to be directly correlated with the antioxidant potential in different in-vitro assays. These polyphenolics are known to possess strong antioxidant properties via mechanisms that may involve direct free- radical scavenging, chelation of metal ions involved in free-radical reactions or an upregulation of the activities of antioxidant enzymes (Asmat et al. 2015).

In this study, different solvents (acetone, aqueous and ethanol) were used for extraction. From the results, all the solvents extracts of the plants used exhibited high polyphenolic content which is correlated with their anti-oxidant activities. The acetone extract of the combination of both plants had the highest phenol and flavonoids content, while proanthocyanidins content was higher in the acetone extract of K. africana and ethanol extract of the combination of both plants had the highest tannins content. Ethanol extract of V. mespilifolia showed a better scavenging potential against ABTS, DPPH and nitric oxide radical while the aqueous extract of the combination of both plants scavenged H2O2 best when compared to other extracts tested.

The pathogenesis of obesity has been linked with an underlying oxidative stress component due to the formation of reactive oxygen species (Savini et al. 2013). Therefore, prevention of

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oxidative stress through the high antioxidant potentials of the two plants and their combination could be one of the ways by which these plants exert their anti-obesity effects.

The increase in multidrug resistance (MDR) in microbial pathogens to conventional drugs has prompted the exploration and development of plant-based products as possible anti-microbial agents (Khan et al. 2009). Moreover, certain organism have associated with the incidence of obesity (Vajro et al. 2013). Therefore, the evaluation of the extracts of Kedrostis africana,

Vernonia mespilifolia and the combination of both plants on selected microbes associated with obesity were carried out. The outcome of the evaluation showed that the extracts possessed has a better potential as anti-fungal agent than as an anti-bacterial agent. Acetone and ethanol extracts of Kedrostis africana, Vernonia mespilifolia and their combination displayed better activities against majority of the tested organisms in comparison to the aqueous extracts which was not active against majority of the tested microbes.

In ethnomedicine, the perception that plants are safe because they are derived from natural sources is totally untrue and deceptive. According to Ekor (2013), the indiscrimate consumption of herbal remedies had caused life-threatening conditions for many consumers.

As a result, the different extracts (acetone, aqueous and ethanol) of Kedrostis africana,

Vernonia mespilifolia and their combination were screened for possible toxicity using both in- vitro (the brine shrimp assay and HeLa cell line) and in-vivo (wistar rat) models. The in-vitro model revealed that only the aqueous and ethanol extracts of K. africana were toxic; while all the three extracts of V. mespilifolia were toxic. The combination of both plants revealed that the aqueous and acetone extracts of the combined plants were toxic in accordance to Meyer’s index of toxicity using brine shrimp. The cytotoxic effect of the aqueous and ethanol extracts of V. mespilifolia, K. africana and their combination were evaluated using HeLa cells. From this study, all the extracts tested had IC50 values were greater than 20 µg/mL which connotes

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that they are not toxic. According to the American National Cancer Institute, crude plant extracts are considered cytotoxic in an in vitro assay when concentrations 20 µg/mL and below produce 50% inhibition of tumor cells, after an exposure time of 48 hours.

In the in-vivo model with the limit where the limit test doses of 2000 and 5000 mg/kg/bwt of aqueous extract of Kedrostis africana, Vernonia mespilifolia and their combination did not produce mortality or significant behavioral changes during 14 days of observation. Also, these extracts administered at doses of 200, 400 and 600 mg/kg/bwt respectively for a period of 28 days did not cause mortality, morbidity or change in relative weight of organs. However, significant weight gain was observed in both treated and control groups. There were no significant alteration in the levels of all the biochemical and haematological parameters investigated. Histopathological examination of the liver and kidney revealed no detectable inflammation. These results indicate that consumption of aqueous extracts of these plants in the doses investigated (200 – 600) mg/kg/bwt can therefore be considered relatively safe with no observed effects on normal growth, liver, kidneys or blood enzymes.

There is a crucial need to developing anti obesity drugs that are efficacious and have minimal side effects (Kang et al. 2013). An important strategy for the management or treatment of obesity could be achieved by developing inhibitor of digestive enzymes (Marrell et al. 2013).

The inhibition of important metabolic enzymes is widely used to determine the anti-obesity ability of potential plant extracts and natural products (Marrell et al. 2013). The present research employed the enzyme-based in-vitro assays to identify potential mechanism and probable anti-obesity actions of the two plants and their combination. Using concentrations ranging from 50 to 200 μg/mL, it was observed that the ethanol extracts of both plant and their combination exhibited weak to moderate inhibitory activities α-amylase, α-glucosidase and pancreatic lipase. This suggests that the anti-obesity properties of the plants were not exerted

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by the inhibition of carbohydrate and fat metabolism but could be by other methods such as thermogenesis and down regulation of adipogenesis.

CONCLUSIONS The studies conducted in the present research demonstrated that:

i. Kedrostis africana and Vernonia mespilifolia despite being considered as wild

plants can still contribute useful amount of nutrients to human and animal diets and

also they had the presence of many macro- and micro nutrients and as such can

serve as a supplement in the prevention of many mineral deficiencies diseases.

ii. The antioxidant properties of K. africana, V. mespilifolia and their combination are

more pronounced in the ethanol and acetone extracts, as demonstrated by their in-

vitro free radical scavenging activities. This observation was corroborated the

relative contents of phenolic acid, flavonoids, proanthocyanidins and tannins, all of

which occurred in larger quantities in the acetone and ethanol extracts.

iii. The antimicrobial properties of Kedrostis africana, Vernonia mespilifolia and their

combination were ascertained. The acetone and ethanol extracts of these plants

inhibited majority of the microbial strain they were tested upon. The fungal strains

were more susceptible to the extracts than the bacterial strains.

iv. The results of this study suggested that the aqueous extracts of both K. africana, V.

mespilifolia and their combination up to the dose of 600 mg/kgb.w. is relatively safe

when administered orally to female and male rats. Since the extracts at the different

doses did not show any lethality, death or adverse effects on the rats, and did not

induce significant alterations in all the biochemical, haematological and

morphological parameters investigated in this study, regardless of gender. Also, the

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aqueous and ethanol extracts of Kedrostis africana, Vernonia mespilifolia and the

combination of both plants were not toxic to HeLa cells. v. At concentration ranging from 50 - 200 μg/ml, the aqueous and ethanol extracts of

both K. africana, V. mespilifolia and their combination exhibited moderate

inhibitory activities against α-amylase, α-glucosidase and lipase. It therefore

appears that the inhibition of these enzymes may not represent a viable anti-obesity

mode of action for the plants.

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Appendix

232

University of Fort Hare Together in Excellence

ETHICAL CLEARANCE CERTIFICATE REC-270710-028-RA Level 01

Certificate Reference Number: AF0051SUNU01

Project title: Assessment of the anti-obesity efficacy and mechanism of action of Kedrostis Africana (L) Cogn and Vernonia mespilifolia Less for the treatment and management of genetically and diet-induced obesity in Wistar rat models.

Nature of Project: PhD

Principal Researcher: Jeremiah Unuofin

Supervisor: Prof A.J Afolayan Co-supervisor: Dr G Otunola

On behalf of the University of Fort Hare's Research Ethics Committee (UREC) I hereby give ethical approval in respect of the undertakings contained in the above mentioned project and research instrument(s). Should any other instruments be used, these require separate authorization. The Researcher may therefore commence with the research as from the date of this certificate, using the reference number indicated above.

Please note that the UREC must be informed immediately of

• Any material change in the conditions or undertakings mentioned in the document • Any material breaches of ethical undertakings or events that impact upon the ethical conduct of the research

232 I I The Principal Researcher must report to the UREC in the prescribed format, where !I applicable, annually, and at the end of the project, in respect of ethical compliance. !)

Special conditions: Research that includes children as per the official regulations of the act must take the following into account: ' Note: The UREC is aware of the provisions of s71 of the National Health Act 61 of 2003 and that matters pertaining to obtaining the Minister's consent are under discussion and remain unresolved. Nonetheless, as was decided at a meeting between the National Health Research Ethics Committee and stakeholders on 6 June 2013, university ethics committees may continue to grant ethical clearance for research involving children without the Minister's consent, provided that the prescripts of the previous rules have been met. This certificate is granted in terms of this agreement.

The UREC retains the right to

• Withdraw or amend this Ethical Clearance Certificate if o Any unethical principal or practices are revealed or suspected o Relevant information has been withheld or misrepresented o Regulatory changes of whatsoever nature so require o The conditions contained in the Certificate have not been adhered to

• Request access to any information or data at any time during the course or after completion of the project. -� . • In addition to the need to comply with the highest level of ethical conduct principle investigators must report back annually as an evaluation and monitoring mechanism on the progress being made by the research. Such a report must be sent to the Dean of Research's office

The Ethics Committee wished you well in your research.

Yours sincerely

Professor John Fisher Mupangwa AREC-Chairperson

16 August 2016

226